WO2013149506A1 - Chimeric peptide based on opioid peptide biphalin and neuropeptide ff and synthesis and use thereof - Google Patents

Chimeric peptide based on opioid peptide biphalin and neuropeptide ff and synthesis and use thereof Download PDF

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WO2013149506A1
WO2013149506A1 PCT/CN2013/070611 CN2013070611W WO2013149506A1 WO 2013149506 A1 WO2013149506 A1 WO 2013149506A1 CN 2013070611 W CN2013070611 W CN 2013070611W WO 2013149506 A1 WO2013149506 A1 WO 2013149506A1
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peptide
resin
opioid
fmoc
analgesic
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PCT/CN2013/070611
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French (fr)
Chinese (zh)
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王锐
方泉
韩政岚
王子龙
李宁
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兰州大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/70—Enkephalins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention belongs to the technical field of biochemistry, and relates to a chimeric peptide constructed based on an opioid peptide and a neuropeptide FF and a method for synthesizing the same; and the invention also relates to the application of the chimeric peptide in the preparation of an analgesic drug with high efficiency and low side effects.
  • opioid peptides in the body can also mediate analgesia by activating opioid receptors.
  • opioid ligand molecules with high selectivity, high efficiency and relatively stable molecules
  • a large number of high-efficiency ligands were chemically modified and screened based on the structure of opioid peptides such as enkephalin.
  • Lipkowski et al. used enkephalin as a chemical template molecule to achieve molecular dimerization by site-directed substitution of two amino acids and C-terminal ⁇ bond of peptide chain.
  • Biphalin A highly efficient opioid peptide Biphalin was successfully constructed, and its chemical structure was (Tyr- OAIa-Gly-Phe-NH) 2 (Pepf/cies 1982, 3:697; Life Sci 1987, 40:2283). Further pharmacological studies have shown that Biphalin can cause strong analgesic effects in both central and intraperitoneal injections, especially in the lateral ventricle, which is 257 times more potent than morphine, and the addictive withdrawal response caused by intraperitoneal injection of Biphalin. Smaller U. Pharmacol. Exp, Ther. 1993, 265: 1446). Therefore, Biphalin has a good application prospect in the development of low-side opioid analgesics.
  • neuropeptides mediate one or more major physiological and pharmacological activities in the body.
  • neuropeptides are accompanied by some secondary activities while exerting their primary activity, and most of the secondary activity has become a side effect.
  • endogenous opioid peptides are accompanied by side effects such as tolerance, addiction, and respiratory depression while relieving pain.
  • the traditional strategy is to use the mother of neuropeptide as a chemical template, and carry out systematic chemical modification to screen out neuropeptide analogs with high receptor selectivity and dominant conformation, so as to maintain high Biological activity and the expected effect of reducing its side effects.
  • Neuropeptide FF was first isolated and identified from bovine brain as an anti-opioid peptide and was found to have anti-morphine analgesic effects (Pmc. Nat. Acad. Sci. USA 1985, 82:7757).
  • the neuropeptide FF system involves two different receptor precursors (Pro-NPFF A and Pro-NPFF B receptors) and different receptors (NPFFi and NPFF 2 receptors) (FEBS Lett. 1997) Mol. Pharmacol. 1999, 55:804; Nat. Cell Biol. 2000, 2: 703; J. Biol. Chem. 2001, 276: 36961; J. Biol. Chem. 2000, 275:25965; 8/ ⁇ ./. Chem.
  • NPFF neuropeptide FF
  • NPFF neuropeptide FF
  • its related peptides can mediate a variety of biological activities.
  • NPFF not only has anti-opioid function, but also has the original opioid activity, which is classified as an important class of opioid regulatory peptides ( ⁇ / ⁇ . Top. Med. Chem. 2005, 5:341).
  • NPFF itself has no effect on the basic pain threshold, that is, it has little effect on the biological activity of the body at physiological dose levels.
  • NPFF as an opioid-regulating peptide, plays an important role in maintaining the balance between endogenous opioid and anti-opioid systems in the body.
  • NPFF neuropeptide
  • Chinese Patent Application No. 201110097843.4 discloses a chimeric peptide based on endomorphin 2 and neuropeptide FF, which utilizes endomorphin 2 on the basis of retaining the N-terminal structure of endomorphin 2 and the C-terminal structure of neuropeptide FF
  • a novel chimeric peptide EN-9 constructed by the C-terminal and phenylalanine residues present in the N-terminus of neuropeptide FF.
  • Pharmacological studies in vivo have found that EN-9 exhibits stronger and more sustained analgesic activity than endomorphin 2, and has the advantages of no tolerance, low addiction and the like.
  • the compound EN-9 subcutaneous EN-9 can cause significant analgesic effects, overcome the defect of endothelin injection without analgesic activity, and thus have potential application value for clinical pain treatment.
  • the analgesic activity of EN-9 is 4-10 times lower than that of the clinical analgesic morphine, resulting in an effective analgesic dose higher than morphine.
  • the object of the present invention is to provide a chimeric peptide BN-9 based on the opioid peptide Biphalin and NPFF with good analgesic effect and low side effects in view of the problems in the prior art.
  • the present invention is based on the opioid peptide Bipha lin and NPFF chimeric peptide (BN-9), which is a chimeric peptide constructed by using the high-efficiency opioid peptide Bipha lin and neuropeptide FF as a chemical template, and the N-terminal is an opioid peptide.
  • BN-9 opioid peptide Bipha lin and NPFF chimeric peptide
  • NPFF opioid peptide
  • the method for synthesizing a chimeric peptide based on the opioid peptide Biphalin and NPFF in the present invention comprises the following process steps:
  • Resin pretreatment Rink-Amide-MBHA resin was stirred in dichloromethane for 30-40 min, the resin was sufficiently swollen, and the solvent was drained under reduced pressure;
  • A/-a-Fmoc protecting group amino acid, ⁇ /-hydroxybenzotriazole, 0-benzotriazole- ⁇ /, ⁇ /, ⁇ /', ⁇ /'- Tetramethylurea-hexafluorophosphate is completely dissolved in DMF, and then mixed with diisopropylethylamine to obtain a mixed solution; then, under the protection of argon, the resin removed by removing the Fmoc group is added to the mixture.
  • reaction was stirred for 40-60 min in the solution, and the solvent was drained; the unreacted A/-a-Fmoc protecting group amino acid, ⁇ /-hydroxybenzotriazole, 0-benzotriazole-oxime were removed by repeated washing with DMF. /, ⁇ /, ⁇ /', ⁇ / '-tetramethylurea-hexafluorophosphate;
  • the amount of hexafluorophosphate is 2-5 times of the molar amount of the resin protected by the Fmoc group;
  • the diisopropylethylamine is used in an amount of 4 to 10 times the molar amount of the resin protected by the Fmoc group;
  • Steps (2), (3) are repeated 8 times, and the amino acids having the /Va-Fmoc protecting group are condensed one by one according to the order of the chimeric peptide structure from the C-terminus to the N-terminus.
  • the resin until the condensation of all amino acid residues is completed, the peptide resin is obtained;
  • cleavage of the peptide chain from the resin completely remove the Fmoc group of the last amino acid of the peptide resin according to the method of the step (2); wash the resin alternately with DCM and MeOH, and thoroughly drain the solvent, and then per gram of the peptide Add 10-25ml resin
  • the cutting agent is cut at room temperature for 1.5-5 hours; filtered, the filtrate is fully dried under reduced pressure at 37 ° C, and then precipitated with diethyl ether not higher than -10 ° C; After the supernatant was removed, the diethyl ether was completely dissolved in water, and the ether phase was removed by a separatory funnel. The aqueous phase was freeze-dried to obtain a white crude solid powder;
  • the dicing agent is a solvent formed by mixing trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5.
  • the product prepared by the above method was detected by mass spectrometry and chromatographic analysis, and was consistent with the designed compound structure. This indicates that the new chimeric peptide of the opioid/neuropeptide FF system has been successfully synthesized, and the purity of the purified sample is above 98%.
  • the invention is based on the opioid peptide Biphalin and NPFF chimeric peptide BN-9, while retaining two key pharmacophores for activating opioid and NPFF receptors, and effectively reducing opioid analgesia while maintaining opioid analgesic effect. Side effects such as tolerance and constipation.
  • the pharmacological function of the chimeric peptide BN-9 of the present invention in terms of pain modulation, tolerance and constipation is demonstrated below by in vivo experiments.
  • mice in the lateral ventricle should be operated in advance to ensure the accuracy of the drug injection site.
  • the lateral ventricle of the mouse was buried using a brain stereotaxic instrument.
  • the head operation area of the mouse was cut off and placed on the brain stereo positioner, and the operation area was disinfected with iodophor.
  • the sagittal suture cuts the opening skin, reveals the skull, and finds the front squat position.
  • mice Spinal cord administration is performed by intrathecal injection in conscious mice, and the specific procedure is described with reference to the methods reported by Hylden and Wilcox (Eur. Pharmacol. 1980, 67: 313). Insert the 25 ⁇ microsampler directly into the subarachnoid space between L5 and L6. When the arachnoid was punctured, the mice had a violent and obvious appendix movement or the tail was "S'' shaped, and saline and drugs were injected into the subarachnoid space at a rate of 5 ⁇ /10 sec.
  • mice were fixed with a fixator before the tail vein injection, exposing the entire tail, and the tail was sprayed with a 75% alcohol cotton ball to spread the veins, and the red veins on both sides of the tail were clearly visible.
  • the injection site is located near 1/3-1/2 of the root of the tail.
  • a lml syringe with a 4 gauge half needle was used with an injection volume of 0.1 ml/piece. Use a left hand to gently straighten the tail when injecting, and a syringe in the right hand to tilt the needle along the tail at an angle of less than 30 degrees. After the needle is inserted, the drug injection is completed within 30 seconds, and after the injection is completed, the cotton ball is pressed to stop bleeding.
  • Kunming male mice were used to detect the effects of drugs on pain sensation using a photothermal appendix.
  • the ambient temperature is controlled at 22 ⁇ 1 °C, and the experimental animals are free to eat and drink.
  • the mouse lateral ventricle was injected with 4 ⁇ of the drug each time.
  • the basic appendix latency (3-5 seconds) was determined prior to dosing, and mice that were too sensitive or dull were discarded.
  • the tail-flick latency of 5, 10, 15, 20, 30, 40, 50, 60, 90 minutes after administration was recorded (the pain detection at the tail vein injection did not measure the tail-flick latency at the 90-minute time point).
  • set the minimum tailing latency to 10 seconds and the tailing time to 10 seconds in 10 seconds.
  • the relevant M PE data were expressed as mean ⁇ standard error (Means ⁇ SEM ). Differences between groups treated with different drug concentrations were analyzed and analyzed by one-way analysis of variance (one-way AN OVA Dunnett test). **P ⁇ 0.001 indicates a significant difference between the M PE value of the drug-treated group and the saline group.
  • the experimental results are shown in Figures 1 ⁇ 3.
  • the analgesic effect of BN-9 and morphine was compared by EC 5Q values.
  • 5 5 values indicate the drug dose at which the drug produces a 50% maximal analgesic effect.
  • BN-9 and morphine were separately mapped in the lateral ventricle, intrathecal and tail vein.
  • the dose-response curve of the M PE value at the maximum analgesic effect time point of the drug level (see Figures 4-6), and their EC 5Q values and 95% confidence intervals at different injection levels were calculated. The relevant data are shown in Table 1. Show.
  • Table 1 Comparison of analgesic effects of different levels of BN-9 and morphine injection a , , EC 50 value (nmol) ( 95% confidence interval) EC 50 ratio administration route 9 « (morphine / BN-9) Ventricular (icv) 0.39 (0.33, 0.46) 1.02 (0.88, 1.18) .61 times Intrathecal (it) 0.29 (0.24, 0.34) 0.35 (0.30, 0.40) 1.21 times tail vein (iv) 58.6 (50.8, 67.4) 46.2 (38.7, 55.2) 0.79 times
  • the saline group was a blank solvent control group, and the concentrations of BN-9 in the lateral ventricle were 0.25, 0.5, 1 and 2 nmol, respectively.
  • the number of animals in each group was 8.
  • the injection of the new compound BN-9 into the lateral ventricle of mice is dose-dependent and has an analgesic effect.
  • the analgesic effect of BN-9 at the level above the spinal cord lasted for 60 minutes and reached the maximum analgesic effect 15 minutes after drug injection.
  • Morphine 0.5, 1, 2, and 4 nmol
  • intrathecal injection of BN-9 (0.125, 0.25, 0.5, 1 and B 2 nmol) can cause a dose-dependent analgesic effect, which is strongest at 10 minutes after drug injection and lasts for 60 minutes (eg Figure 2).
  • the morphine (0.125, 0.25, 0.5, 1 and 2 nmol) injected horizontally on the spinal cord only lasted for 40 minutes, and it caused the greatest analgesic effect 10 minutes after the injection.
  • the effect is 60 minutes (as shown in Figure 3).
  • the analgesic activity caused by tail vein injection of morphine (0.25, 0.5, 1 and 2 mg) was also sustained for 60 minutes, and the maximum analgesic effect time was 10 minutes after drug injection.
  • mice were injected with the drug for eight consecutive days in the lateral ventricle of the mice, and their analgesic tolerance was examined by photothermal appendix method, and the pharmacological activity of the compound of the present invention BN-9 and morphine in analgesic tolerance was further compared.
  • mice Male Kunming mice were injected intracerebroventricularly or intrathecally for eight days, once a day, using a photothermal appendix to detect the effect of continuous drug injection on pain perception.
  • the ambient temperature is controlled at 22 ⁇ 1 ° C, and the experimental animals are free to eat and drink.
  • the mouse lateral ventricle was injected with 4 ⁇ of the drug each time.
  • the baseline tail-flick latency (3-5 seconds) was measured before the first day of drug injection, and mice that were too sensitive or dull were discarded.
  • the tail time latency of the maximum analgesic effect time of the relevant drug after administration was recorded in the experiment, such as the recording time points of the lateral ventricle injection of ⁇ -9 and morphine were 15 and 30 minutes after administration, respectively; intrathecal injection of these two drugs Recording time points were given to the drug for 10 minutes; the time of the blank solvent control group was recorded with ⁇ -9 be consistent. To prevent burns on the tail of the mouse, set the maximum tailing latency to 10 seconds.
  • the analgesic tolerance of the drug was compared using the maximum analgesic effect of the drug at the time point of the sputum.
  • ⁇ Data were expressed as mean ⁇ standard error (Means ⁇ SEM). Differences in eight-day analgesic effects in mice were statistically analyzed by one-way ANOVA (one-way AN0VA Bonferroni test), ***P ⁇ 0.001 indicates There was a significant difference in the analgesic effect of morphine on the first day.
  • the experimental results are shown in Figures 7 and 8.
  • the saline group was a blank solvent control group, the dose of BN-9 in the lateral ventricle was 0.5, 1, 2 and 4 nmol, and the dose of morphine was 4 nmol.
  • the experimental data in Figure 7 showed that the lateral ventricle of the mice was injected for eight consecutive days. After the solvent was compared with physiological saline, the mice did not produce analgesic activity. Compared with the blank solvent control saline group, the intraventricular injection of morphine and various doses of BN-9 on the first day significantly increased the tail-flick latency of the mice. However, in the lateral ventricle of mice, the morphine group was continuously injected for eight days.
  • opioid analgesics can cause side effects such as constipation. Therefore, the role of the chimeric peptide BN-9 and morphine in the regulation of gastrointestinal motility was further evaluated. Drugs were injected into the lateral ventricles of mice, and their effects on gastrointestinal motility were evaluated by the toner gastrointestinal motility assay.
  • Kunming male mice weighing 20 ⁇ 2 g.
  • the lateral ventricle was restored to the gastrointestinal motion test after 4 days of recovery.
  • the commonly used toner detection method is used for the detection of body gastrointestinal motility (Peptides, 2000, 21:295).
  • the specific experimental procedure is as follows: Before the gastrointestinal exercise experiment, the mice were fasted for 16 hours (free drinking during fasting), then the side ventricle was injected with drugs, and the activated carbon suspension prepared in advance after 15 minutes (a 5% activated carbon) And a physiological saline suspension of 10% gum arabic) was orally infused into the stomach in a volume of 0.1 ml per 10 g of body weight.
  • Gastrointestinal exercise test results were evaluated as a percentage of gastrointestinal motility, and the percentage of gastrointestinal motility per mouse was calculated by dividing the moving distance of the activated carbon suspension by the percentage after the total length of the small intestine.
  • the relevant data were expressed as mean ⁇ standard error (Mea nS ⁇ SEM) of the percentage of gastrointestinal motility, and differences between groups treated with different drug concentrations were statistically analyzed by one-way analysis of variance (one-way AN OVA Bonferroni test). Analysis, ⁇ 0.01, ***P ⁇ 0.001 indicates a significant difference between the percentage of gastrointestinal motility in the drug-treated group and the saline group.
  • the experimental results are shown in Figure 9.
  • the saline group was a blank solvent control group
  • the morphine concentration was 4 nmol
  • the BN-9 drug concentrations were 0.5, 1, 2, and 4 n m O l , respectively.
  • the number of mice in each group was 9.
  • the experimental results showed that the percentage of gastrointestinal motility in the blank solvent control group was 80%.
  • the morphine group injected into the lateral ventricle 4 nmol significantly inhibited gastrointestinal motility in mice, and the percentage of gastrointestinal motility was only 14%. .
  • Kunming male mice weighing 20 ⁇ 2 g. On the 5th day after the lateral ventricle was buried, it was used for the formalin-induced pain test. The ambient temperature is controlled at 22 ⁇ 1 ° C, and the experimental animals are free to eat and drink. Refer to Curr. Protoc. Neurosci. ( 8.9.1-8.9.16.), published by John Wiley & Sons, Inc. 2007, for specific test methods. The specific experimental procedure was as follows: the mice were placed in a plexiglass box (20 x 20 x 30 cm), first adapted to the environment for 10-15 minutes, and then the mice were injected with the drug into the lateral ventricle at a dose of 5 ⁇ .
  • mice After 5 minutes of administration, the mice were taken out, and the skin of the neck and back of the mouse was gently grasped by hand, the little finger was pressed against the tail of the mouse, and the mouse was fixed with the abdomen facing upward to expose the hind paw of the mouse.
  • a 20 ⁇ M formalin solution (5 % concentration) was injected subcutaneously into the skin of the right hind paw of the mouse, and the mice were quickly returned to the observation box, and timing was started, and 40 minutes after the injection of formalin solution was recorded.
  • Mouse behavioral response within minutes. Formalin generally causes two phases of response. According to the experimental needs, two behavioral recording methods were used: (1) The mice were injected, bitten, and shaken every 1 minute after injection of formalin. A total of 40 minutes of recording time; (2) The time to inject the foot in the mouse, bite, and shake was recorded within 0 to 5 minutes (first phase) and 15-30 minutes (second phase) after injection of formalin.
  • the saline group was a blank solvent control group
  • the BN-9 and morphine treatment groups had a drug concentration of 5 and 10 nmoL, respectively, and the number of animals per group was 7 to 10.
  • the experimental results showed that formalin caused two pain sensations after the injection of normal saline into the lateral ventricle, ie, the acute phase reaction of the first phase from 0 to 5 minutes and the second phase inflammation pain of 15 to 30 minutes. reaction. Injection of 5 nmol of BN-9 and 10 nmol of morphine into the lateral ventricle significantly inhibited the two-phase pain response induced by formalin.
  • the experimental results show that different doses of BN-9 and morphine have strong analgesic activity on the first phase of acute pain, and the inhibition rate is between 70 and 100%.
  • the dose-response curve showed that BN-9 had a slightly stronger analgesic effect on the second phase of inflammatory pain than morphine.
  • the chimeric peptide BN-9 based on the opioid peptide Biphalin and NPFF exhibits stronger central analgesic activity than morphine, and has the advantages of no analgesic tolerance and little effect on gastrointestinal motility, and is effective. Overcoming side effects such as tolerance and constipation, which are common in opioid analgesics.
  • intracerebroventricular injection of BN-9 significantly inhibited the acute pain and inflammatory pain caused by formalin, and the analgesic effect was slightly stronger than morphine, so the chimeric peptide BN-9 has potential application value in the treatment of clinical pain. .
  • Figure 1 is a time-effect curve of dose-dependent analgesia produced by injection of BN-9 into the lateral ventricle of mice;
  • Figure 2 is a time-effect curve of dose-dependent analgesic effect produced by intrathecal injection of BN-9 in mice
  • Figure 3 is a time-effect curve of dose-dependent analgesic effects produced by tail vein injection of BN-9 in mice.
  • A for injection
  • B for injection
  • Figure 4 is a dose-response curve of the analgesic effect of BN-9 and morphine injected into the lateral ventricle of mice;
  • Figure 5 is a dose-response curve of the analgesic effect of intrathecal injection of BN-9 and morphine in mice;
  • Figure 6 is a dose-response curve of the analgesic effect of BN-9 and morphine injected into the tail vein of mice;
  • Figure 7 shows the changes in analgesic effects caused by intracerebroventricular injection of BN-9 and morphine for eight consecutive days in the spinal cord and spinal cord of mice;
  • Figure 8 shows the changes in analgesic effects caused by intrathecal injection of BN-9 and morphine for eight consecutive days in the spinal cord and spinal cord of mice;
  • Figure 11 is a dose-effect curve of acute pain (first phase) inhibition by formalin injection of BN-9 and morphine in formalin;
  • Figure 12 is a dose-effect curve of inflammatory pain (second phase) inhibition by formalin injection of BN-9 and morphine, respectively.
  • High performance liquid chromatography was Waters' Delta 600; analytical column: DELTA PAK 5 ⁇ [18 300A 3.9xl50mm ; preparative column: DELTA PAK 15 ⁇ C18 300 ⁇ 7.8x300mm.
  • the mass spectrometer was PE Biosystems, Mariner System 5074.
  • the manual solid phase peptide synthesizer was designed by the laboratory and made by the glassworker (the design principle of the synthesizer is specifically referred to the "Fmoc solid phase peptide synthesis" compiled by Chen WC and White PD), and is shown in Fig. 4 on page 14
  • the mechanical stirring method is used instead of the drum nitrogen method to achieve the purpose of thorough mixing of the reaction solution.
  • A/-a-Fmoc protected amino acid (Fmoc-Aa), ⁇ /-hydroxybenzotriazole (HOBt), 0-benzotriazole- ⁇ /, ⁇ /, ⁇ /', ⁇ / '- Tetramethylurea-hexafluorophosphate (HBTU), diisopropylethylamine (DI EA) and triisopropylsilane (TIS) were purchased from Jill Biochemical (Shanghai) Co., Ltd.
  • Ninhydrin is a product of Shanghai Reagent III.
  • Dichloromethane (DCM), hydrazine/, hydrazine/-dimethylformamide (DMF), hexahydropyridine (piperidine), methanol (MeOH) and pyridine were purchased from Tianjin Second Reagent Factory, trifluoroacetic acid (TFA). ), phenol and pyridine are all Tianjin reagents Factory products; The above organic reagents are re-steamed before use.
  • Resin pretreatment 500 mg of Rink-Amide-MBHA resin was added to the synthesizer, and 8 ml of DCM was added and stirred for 30 min to fully swell the resin and then drain the solvent under reduced pressure.
  • the Fmoc group of the last amino acid of the peptide resin is completely removed according to the procedure in the above step (2). Then, according to DCM 3 min x 2 times, MeOH 3 min x 1 time; DCM 3 min x 1 times, MeOH 3 min x 2 times alternately wash the resin. Remove the stir bar, seal the synthesizer (gel plug), and drain thoroughly (at least 2 hours).
  • the whole crude product was dissolved in a 20% acetic acid solution, and the solution was passed through a Sephadex G25 cross-linked Sephadex column (2.0 x 25 cm), and the mobile phase was a 20% acetic acid solution.
  • the main peak was collected by an ultraviolet detector, and then freeze-dried to obtain 116.2 mg of a white powder after desalting.
  • the above-mentioned desalted peptide compound was separated and purified by reverse-phase high performance liquid chromatography (HPLC) C 18 column (XBridgeTM BEH 130 C 18 , 19 mm ⁇ 250 mm). After separation, the main peak of the sample was collected and lyophilized to obtain a white pure peptide solid. Powder 82.2 mg o
  • the purity of the purified BN-9 sample was above 98%, and the total yield of the synthesis was 40%.
  • the results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • System 1 Gradient elution system 1 is: 10-100% acetonitrile/water (0.05% TFA) (completed in 30 minutes), flow rate: l mL/min, detection wavelength 220 nm Analytical column: Delta Pak C 18 , 5 ⁇ , 150 ⁇ 3.9 mm ;
  • System 2 Gradient elution system 2: 10-80% acetonitrile/water (0.05% TFA) (completed in 30 minutes), flow rate: 1 mL/min, detection wavelength is 220 nm, analytical column: Delta Pak C 18 , 5 ⁇ , 150 x 3.9 mm.

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Abstract

Provided in the present invention is a chimeric peptide based on opioid peptide Biphalin and NPFF. Also provided in the present invention are the synthesis method for the chimeric peptide and the use thereof in preparing analgesic drugs. The chimeric peptide of the present invention has stronger central analgesic activity than morphine, and has the advantages of no analgesic tolerance, little effect on gastrointestinal motility, etc.

Description

基于阿片肽 Biphalin和神经肽 FF的嵌合肽及其合成和应用  Chimeric peptide based on opioid peptide Biphalin and neuropeptide FF and its synthesis and application
技术领域 Technical field
本发明属于生化技术领域， 涉及一种基于阿片肽和神经肽 FF而构建的嵌合肽及其合 成方法； 本发明同时还涉及该嵌合肽在高效、 低副作用镇痛药物制备中的应用。  The invention belongs to the technical field of biochemistry, and relates to a chimeric peptide constructed based on an opioid peptide and a neuropeptide FF and a method for synthesizing the same; and the invention also relates to the application of the chimeric peptide in the preparation of an analgesic drug with high efficiency and low side effects.
背景技术 Background technique
疼痛是人类所经受的最常见的病症之一， 每个人的一生都会经历疼痛的困扰。据统计 全球大约有 1/3的人忍受着持续或周期性疼痛的折磨， 美国在疼痛相关社会问题的花费约 为 1千亿美元 /年 ( Proc. Nat. Acad. Sci. U.S.A. 2001, 98: 11845)。 在临床上， ***类的阿片 药物已被人们广泛的应用于治疗和缓解各种疼痛，但由于阿片类的镇痛药物能引起严重的 耐受、 成瘾、 便秘和呼吸抑制等不良反应， 从而大大限制了其临床应用范围。  Pain is one of the most common conditions that humans experience, and everyone experiences pain throughout their lives. According to statistics, about one-third of the world suffers from persistent or periodic pain, and the cost of pain-related social problems in the United States is about $100 billion/year (Proc. Nat. Acad. Sci. USA 2001, 98: 11845). Clinically, morphine opioids have been widely used to treat and alleviate various types of pain, but because opioid analgesics can cause serious adverse reactions such as tolerance, addiction, constipation and respiratory depression, It greatly limits the scope of its clinical application.
已有的研究结果表明， 机体内的阿片肽同样也能通过激活阿片受体而介导镇痛作用。 为了获得选择性高、 高效、 分子较为稳定的阿片类配体分子， 在脑啡肽等阿片肽的结构基 础上通过化学修饰并筛选出了大量的高效配体。 Lipkowski等人以脑啡肽为化学模板分子， 通过 2位氨基酸定点替换、肽链 C末端肼键连接实现了分子二聚化， 成功构建了一个高效 的阿片肽 Biphalin ,其化学结构为 (Tyr-OAIa-Gly-Phe-NH)2 ( Pepf/cies 1982, 3 :697； Life Sci 1987, 40:2283)。 进一步的药理学活性研究表明， Biphalin 在中枢和腹腔注射时都能引起强镇痛 作用， 特别是侧脑室注射时其镇痛强度为***的 257倍， 并且腹腔注射 Biphalin引起的成 瘾戒断反应较小 U. Pharmacol. Exp, Ther. 1993, 265:1446)。 因此， Biphalin在低副作用的阿 片类镇痛药的研发中具有较好的应用前景。 Previous studies have shown that opioid peptides in the body can also mediate analgesia by activating opioid receptors. In order to obtain opioid ligand molecules with high selectivity, high efficiency and relatively stable molecules, a large number of high-efficiency ligands were chemically modified and screened based on the structure of opioid peptides such as enkephalin. Lipkowski et al. used enkephalin as a chemical template molecule to achieve molecular dimerization by site-directed substitution of two amino acids and C-terminal 肼 bond of peptide chain. A highly efficient opioid peptide Biphalin was successfully constructed, and its chemical structure was (Tyr- OAIa-Gly-Phe-NH) 2 (Pepf/cies 1982, 3:697; Life Sci 1987, 40:2283). Further pharmacological studies have shown that Biphalin can cause strong analgesic effects in both central and intraperitoneal injections, especially in the lateral ventricle, which is 257 times more potent than morphine, and the addictive withdrawal response caused by intraperitoneal injection of Biphalin. Smaller U. Pharmacol. Exp, Ther. 1993, 265: 1446). Therefore, Biphalin has a good application prospect in the development of low-side opioid analgesics.
众所周知， 神经肽在机体内都能介导一种或多种主要的生理和药理学活性。 但是， 神 经肽在发挥其主要活性的同时也伴随着一些次要活性，而大部分次要活性成为了它的副作 用。 例如， 内源性阿片肽在缓解疼痛的同时， 会伴随着耐受、 成瘾和呼吸抑制等副作用。 为了解决这一矛盾问题， 传统的策略是以神经肽的母体为化学模板， 进行了***的化学修 饰而筛选出受体选择性较高且具有优势构象的神经肽类似物，从而达到保持较高生物活性 并降低其副作用的预期效果。  It is well known that neuropeptides mediate one or more major physiological and pharmacological activities in the body. However, neuropeptides are accompanied by some secondary activities while exerting their primary activity, and most of the secondary activity has become a side effect. For example, endogenous opioid peptides are accompanied by side effects such as tolerance, addiction, and respiratory depression while relieving pain. In order to solve this contradiction problem, the traditional strategy is to use the mother of neuropeptide as a chemical template, and carry out systematic chemical modification to screen out neuropeptide analogs with high receptor selectivity and dominant conformation, so as to maintain high Biological activity and the expected effect of reducing its side effects.
近年来还发展了一些新策略， 即基于高效的肽类配体设计并合成了一系列嵌合肽， 以 期使嵌合肽在发挥主要活性的同时， 能最大限度的降低其副作用。 尤其是， Foran等人基 于阿片肽 EM-2 和速激肽 P 物质 （SP ) 而成功地设计构建了一种全新的杂合肽 ESP7 (Tyr-Pro-Phe-Phe-Gly-Leu-Met- NH2)，并且发现脊髓水平注射 ESP7能产生阿片受体依赖的 镇痛作用，但又不产生镇痛耐受的阿片样副作用（Prac. Nat. Acad. Sci. U.S.A. 2000, 97:7621)。 神经肽 FF最早是作为一种抗阿片肽从牛脑中被分离和鉴定的， 并发现它具有抗*** 镇痛的作用 ( Pmc. Nat. Acad. Sci. U.S.A. 1985, 82:7757)。 神经肽 FF***涉及到两种不同的 受体前体（Pro-NPFFA禾口 Pro-NPFFB受体）禾口两禾中不同受体（ NPFFi禾口 NPFF2受体） ( FEBS Lett. 1997, 409:426； Mol. Pharmacol. 1999, 55:804； Nat. Cell Biol. 2000, 2: 703； J. Biol. Chem. 2001, 276:36961； J. Biol. Chem. 2000, 275:25965； 8/·。/. Chem. 2000, 275 :39324等）。 近年的药理 学活性鉴定表明， NPFF及其相关肽能介导多种生物学活性， NPFF不仅具有抗阿片的功能， 还具有原阿片活性，从而被归为一类重要的阿片调节肽（ ν/τ. Top. Med. Chem. 2005, 5:341)。 NPFF自身对基础痛阈值无影响，即在生理剂量水平对机体的生物学活性影响不大。但 NPFF 作为阿片调节肽， 在维持机体的内源性阿片和抗阿片***平衡过程中起着重要作用， 它参 与了镇痛耐受的调节， 并能减弱***等阿片物质所引起的条件位置偏爱作用 ( CU/T. Top. Med. Chem. 2005, 5 :341； Peptides. 2006, 27:964； Peptides. 2007, 28:2235； Peptides. 2010, 31:1374等）。 因此， NPFF 的药理学功能使其适合作为化学模板分子来发展一类全新的阿 片 /NPFF***的嵌合肽。 In recent years, some new strategies have been developed to design and synthesize a series of chimeric peptides based on highly efficient peptide ligands, in order to maximize the side effects of chimeric peptides while exerting their main activities. In particular, Foran et al. successfully designed and constructed a novel hybrid peptide ESP7 based on opioid peptide EM-2 and tachykinin substance P (SP) (Tyr-Pro-Phe-Phe-Gly-Leu-Met- NH 2 ), and injection of ESP7 at the spinal cord level was found to produce opioid receptor-dependent analgesic effects without the production of analgesic tolerance opioid side effects (Prac. Nat. Acad. Sci. USA 2000, 97:7621). Neuropeptide FF was first isolated and identified from bovine brain as an anti-opioid peptide and was found to have anti-morphine analgesic effects (Pmc. Nat. Acad. Sci. USA 1985, 82:7757). The neuropeptide FF system involves two different receptor precursors (Pro-NPFF A and Pro-NPFF B receptors) and different receptors (NPFFi and NPFF 2 receptors) (FEBS Lett. 1997) Mol. Pharmacol. 1999, 55:804; Nat. Cell Biol. 2000, 2: 703; J. Biol. Chem. 2001, 276: 36961; J. Biol. Chem. 2000, 275:25965; 8/·./. Chem. 2000, 275: 39324, etc.). In recent years, pharmacological activity identification has shown that NPFF and its related peptides can mediate a variety of biological activities. NPFF not only has anti-opioid function, but also has the original opioid activity, which is classified as an important class of opioid regulatory peptides (ν/ τ. Top. Med. Chem. 2005, 5:341). NPFF itself has no effect on the basic pain threshold, that is, it has little effect on the biological activity of the body at physiological dose levels. However, NPFF, as an opioid-regulating peptide, plays an important role in maintaining the balance between endogenous opioid and anti-opioid systems in the body. It participates in the regulation of analgesic tolerance and can attenuate the conditional positional preference caused by opioids such as morphine. Role (CU/T. Top. Med. Chem. 2005, 5:341; Peptides. 2006, 27:964; Peptides. 2007, 28:2235; Peptides. 2010, 31:1374, etc.). Therefore, the pharmacological function of NPFF makes it suitable as a chemical template molecule to develop a novel class of chimeric peptides for the opioid/NPFF system.
中专利申请 201110097843.4公开了一种基于内***肽 2和神经肽 FF的嵌合肽， 其在 保留内***肽 2的 N末端结构和神经肽 FF的 C末端结构基础上， 利用内***肽 2的 C末 端和神经肽 FF的 N末端中都存在的苯丙氨酸残基的特点而构建的一种全新的嵌合肽 EN-9。 体内的药理学研究发现： EN-9表现出比内***肽 2更强、更持续的镇痛活性， 并具有无耐 受作用、 低成瘾性等优点。 此外， 化合物 EN-9皮下 EN-9能引起显著的镇痛作用， 能克服 内***肽外周注射无镇痛活性的缺陷，从而具有用于临床疼痛治疗的潜在应用价值。但是， EN-9的镇痛活性比临床用镇痛药***低 4-10倍， 从而导致其有效镇痛剂量高于***。 发明内容  Chinese Patent Application No. 201110097843.4 discloses a chimeric peptide based on endomorphin 2 and neuropeptide FF, which utilizes endomorphin 2 on the basis of retaining the N-terminal structure of endomorphin 2 and the C-terminal structure of neuropeptide FF A novel chimeric peptide EN-9 constructed by the C-terminal and phenylalanine residues present in the N-terminus of neuropeptide FF. Pharmacological studies in vivo have found that EN-9 exhibits stronger and more sustained analgesic activity than endomorphin 2, and has the advantages of no tolerance, low addiction and the like. In addition, the compound EN-9 subcutaneous EN-9 can cause significant analgesic effects, overcome the defect of endothelin injection without analgesic activity, and thus have potential application value for clinical pain treatment. However, the analgesic activity of EN-9 is 4-10 times lower than that of the clinical analgesic morphine, resulting in an effective analgesic dose higher than morphine. Summary of the invention
本发明的目的是针对现有技术中存在的问题， 提供一种镇痛效果好、 副作用低的基于 阿片肽 Biphalin和 NPFF的嵌合肽 BN-9。  The object of the present invention is to provide a chimeric peptide BN-9 based on the opioid peptide Biphalin and NPFF with good analgesic effect and low side effects in view of the problems in the prior art.
本发明的另一目的是提供一种基于阿片肽 Bipha lin和 NPFF的嵌合肽 BN-9的合成方法。 本发明还有一个目的， 就是提供该基于阿片肽 Bipha lin和 NPFF的嵌合肽 BN-9在制备 镇痛药物中的应用。  Another object of the present invention is to provide a method for synthesizing the chimeric peptide BN-9 based on the opioid peptides Bipha lin and NPFF. Still another object of the present invention is to provide the use of the opioid peptide Biphalin and NPFF-based chimeric peptide BN-9 for the preparation of analgesics.
(一） 基于阿片肽 Biphalin和 NPFF的嵌合肽  (i) Chimeric peptides based on opioid peptides Biphalin and NPFF
本发明基于阿片肽 Bipha lin和 NPFF的嵌合肽 （BN-9 ) , 是以高效阿片肽 Bipha lin和神 经肽 FF为化学模板，构建的嵌合肽，其 N末端为阿片肽 "药效团"（即半个分子的 Biphalin) , C末端为 NPFF "药效团 "（即 NPFF的 C端五肽结构， NPFF ( 3-8 ) )， 其结构式如下：
Figure imgf000004_0001
The present invention is based on the opioid peptide Bipha lin and NPFF chimeric peptide (BN-9), which is a chimeric peptide constructed by using the high-efficiency opioid peptide Bipha lin and neuropeptide FF as a chemical template, and the N-terminal is an opioid peptide. "(ie half-molecule Biphalin), the C-terminus is NPFF "pharmacophore" (ie C-terminal pentapeptide structure of NPFF, NPFF (3-8)), and its structural formula is as follows:
Figure imgf000004_0001
(二） 基于阿片肽 Biphalin和 NPFF的嵌合肽的合成 (II) Synthesis of chimeric peptides based on opioid peptides Biphalin and NPFF
本发明中基于阿片肽 Biphalin和 NPFF的嵌合肽的合成方法， 包括以下工艺步骤： The method for synthesizing a chimeric peptide based on the opioid peptide Biphalin and NPFF in the present invention comprises the following process steps:
( 1) 树脂预处理： 将 Rink-Amide-MBHA树脂在二氯甲烷中搅拌 30-40 min, 使树脂充 分溶胀后减压抽干溶剂； (1) Resin pretreatment: Rink-Amide-MBHA resin was stirred in dichloromethane for 30-40 min, the resin was sufficiently swollen, and the solvent was drained under reduced pressure;
( 2)脱除 Fmoc保护：将溶胀、抽干溶剂后的树脂在体积浓度 18-25 %的六氢吡啶 /DMF 溶液中， 搅拌 3-10min后抽干， 重复 2-3次； 再加入体积浓度 18-25 %的六氢吡啶 /DMF溶 液， 搅拌 10-15min， 使 Fmoc基团脱除完全， 然后抽干溶剂； 最后用 DMF洗涤除净六氢 吡啶， 得到脱除 Fmoc基团保护的树脂；  (2) Removal of Fmoc protection: the resin after swelling and drying the solvent is stirred in a volume concentration of 18-25% in a solution of hexahydropyridine/DMF, stirred for 3-10 minutes, then drained, and repeated 2-3 times; The solution of 18-25% hexahydropyridine/DMF was stirred for 10-15 min to completely remove the Fmoc group, and then the solvent was drained. Finally, the dihydropyridine was removed by washing with DMF to obtain a resin which was protected by Fmoc group. ;
( 3 ) 縮合： 依次将 A/-a-Fmoc 保护基团氨基酸、 Λ/-羟基苯并三氮唑、 0-苯并三氮唑 -Λ/,Λ/,Λ/',Λ/'-四甲基脲-六氟磷酸盐完全溶解于 DMF中，再加入二异丙基乙胺后混匀得混合溶 液； 然后在氩气保护下， 将脱除 Fmoc 基团保护的树脂加入所述混合溶液中搅拌反应 40-60min， 抽干溶剂； 用 DMF重复洗涤除去未反应的 A/-a-Fmoc保护基团氨基酸、 Λ/-羟基 苯并三氮唑、 0-苯并三氮唑 -Λ/,Λ/,Λ/',Λ/ '-四甲基脲-六氟磷酸盐；  (3) Condensation: A/-a-Fmoc protecting group amino acid, Λ/-hydroxybenzotriazole, 0-benzotriazole-Λ/, Λ/, Λ/', Λ/'- Tetramethylurea-hexafluorophosphate is completely dissolved in DMF, and then mixed with diisopropylethylamine to obtain a mixed solution; then, under the protection of argon, the resin removed by removing the Fmoc group is added to the mixture. The reaction was stirred for 40-60 min in the solution, and the solvent was drained; the unreacted A/-a-Fmoc protecting group amino acid, Λ/-hydroxybenzotriazole, 0-benzotriazole-oxime were removed by repeated washing with DMF. /,Λ/,Λ/',Λ/ '-tetramethylurea-hexafluorophosphate;
所述 A/-a-Fmoc保护基团氨基酸、 Λ/-羟基苯并三氮唑、 0-苯并三氮唑 -Λ/,Λ/,Λ/',Λ/'-四甲基 脲-六氟磷酸盐的用量分别为脱除 Fmoc基团保护的树脂摩尔量的 2-5倍；  The A/-a-Fmoc protecting group amino acid, Λ/-hydroxybenzotriazole, 0-benzotriazole-oxime/, Λ/, Λ/', Λ/'-tetramethylurea- The amount of hexafluorophosphate is 2-5 times of the molar amount of the resin protected by the Fmoc group;
所述二异丙基乙胺的用量为脱除 Fmoc基团保护的树脂摩尔量的 4-10倍；  The diisopropylethylamine is used in an amount of 4 to 10 times the molar amount of the resin protected by the Fmoc group;
(4)肽链的延长： 重复步骤（2)、 ( 3 ) 8次， 按照嵌合肽结构由 C-末端向 N-末端的顺 序， 依次将带有 /V-a-Fmoc保护基团氨基酸逐个縮合至树脂上， 直至完成所有氨基酸残基 的縮合， 得到肽树脂；  (4) Extension of the peptide chain: Steps (2), (3) are repeated 8 times, and the amino acids having the /Va-Fmoc protecting group are condensed one by one according to the order of the chimeric peptide structure from the C-terminus to the N-terminus. To the resin, until the condensation of all amino acid residues is completed, the peptide resin is obtained;
( 5 ) 肽链从树脂上的切割： 按照步骤 （2 ) 的方法将肽树脂最后一个氨基酸的 Fmoc 基团完全脱除；用 DCM、MeOH交替洗涤树脂，充分抽干溶剂后，按每克肽树脂加入 10-25ml 的切割剂， 于室温下切割反应 1.5-5小时； 过滤， 滤液在不高于 37°C的条件下充分减压旋 干， 然后用不高于 -10°C的***中析出沉淀； 静置后先去除上清的***， 再用水充分溶解 后用分液漏斗除去***相， 水相经冷冻干燥， 得白色粗肽固体粉末； (5) cleavage of the peptide chain from the resin: completely remove the Fmoc group of the last amino acid of the peptide resin according to the method of the step (2); wash the resin alternately with DCM and MeOH, and thoroughly drain the solvent, and then per gram of the peptide Add 10-25ml resin The cutting agent is cut at room temperature for 1.5-5 hours; filtered, the filtrate is fully dried under reduced pressure at 37 ° C, and then precipitated with diethyl ether not higher than -10 ° C; After the supernatant was removed, the diethyl ether was completely dissolved in water, and the ether phase was removed by a separatory funnel. The aqueous phase was freeze-dried to obtain a white crude solid powder;
所述切割剂是由三氟乙酸、三异丙基硅烷、水以 95:2.5:2.5的体积比混合形成的溶剂。 ( 6 ) 粗肽的脱盐和纯化： 以体积浓度 10-20%的乙酸溶液为流动相， 将粗肽通过 SePhadeX G25交联葡聚糖凝胶柱脱盐， 利用紫外检测仪收集主峰后冷冻干燥， 得到脱盐的 肽化合物； 再利用反向高效液相色谱柱进行分离纯化， 收集主峰， 冷冻干燥后得到白色的 纯肽固体粉末。 The dicing agent is a solvent formed by mixing trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5. (6) Desalination and purification of crude peptide: The crude peptide was desalted by Se P had eX G25 cross-linked Sephadex column using a 10-20% acetic acid solution as a mobile phase, and the main peak was collected by UV detector. Freeze-drying to obtain a desalted peptide compound; separation and purification by reverse-phase high performance liquid chromatography column, main peaks were collected, and lyophilized to obtain a white pure peptide solid powder.
上述方法制备的产物经质谱和色谱分析检测， 与设计的化合物结构一致。表明成功合 成了阿片 /神经肽 FF***的全新嵌合肽， 纯化后样品纯度在 98 %以上。  The product prepared by the above method was detected by mass spectrometry and chromatographic analysis, and was consistent with the designed compound structure. This indicates that the new chimeric peptide of the opioid/neuropeptide FF system has been successfully synthesized, and the purity of the purified sample is above 98%.
(三） 基于阿片肽 Biphalin和 NPFF的嵌合肽的活性实验  (iii) Activity assay of chimeric peptides based on opioid peptides Biphalin and NPFF
本发明基于阿片肽 Biphalin和 NPFF的嵌合肽 BN-9,同时保留了激活阿片和 NPFF受体 的两个关键药效团， 在保持了阿片镇痛作用的同时， 能有效地降低阿片镇痛耐受和便秘等 副作用。 下面通过体内实验说明本发明的嵌合肽 BN-9在痛觉调节、 耐受和便秘方面的药 理学功能。  The invention is based on the opioid peptide Biphalin and NPFF chimeric peptide BN-9, while retaining two key pharmacophores for activating opioid and NPFF receptors, and effectively reducing opioid analgesia while maintaining opioid analgesic effect. Side effects such as tolerance and constipation. The pharmacological function of the chimeric peptide BN-9 of the present invention in terms of pain modulation, tolerance and constipation is demonstrated below by in vivo experiments.
1、 BN-9的镇痛实验  1. Analgesic experiment of BN-9
通过侧脑室、脊髓和尾静脉三个水平注射后， 利用小鼠光热甩尾实验进行了体内痛觉 调节作用的活性检测和比较。 并以阳性对照药物***作为对照组。  After three levels of injection into the lateral ventricle, spinal cord and tail vein, the activity of in vivo pain modulation was detected and compared using the mouse photothermal tail-flick test. The positive control drug morphine was used as a control group.
( 1) 小鼠的侧脑室给药  (1) Lateral ventricle administration in mice
侧脑室水平痛觉检测实验用小鼠， 需提前进行小鼠的侧脑室埋管手术， 以保证药物注 射位点的准确性。 利用脑立体定位仪进行小鼠的侧脑室埋管。 昆明系雄性小鼠， 体重 20±2 g; 脑立体定位仪为江湾 I型。 小鼠用戊巴比妥钠麻醉 （i.p， 80mg/kg小鼠体重） 后， 将小 鼠头部手术区剪去毛发， 置于脑立体定位仪上固定后， 手术区用碘伏消毒， 沿矢状缝切开 头皮， 露出颅骨， 找到前囟位置。 从前囟向后 3 mm， 向左 /右 l mm， 即为侧脑室的上方位 置。 自制不锈钢管（24-gauge的一段， 上面接一小段 PE-10管）按上述位置向下*** 3 mm， 即为侧脑室。 一段不锈钢琴弦 （28-gauge ) ***到上述钢管中， 以防止脑脊液外溢、 感染 和堵塞钢管。用医科牙托粉和胶水固定钢管， 凝固后， 缝合伤口。手术后， 小鼠恢复 4天， 第 5天开始后续实验。 实验中涉及到的手术器械、 不锈钢管等均已在手术前消毒。 药物溶 解在生理盐水中， 在 -20°C保存， 使用前解冻。  In the lateral ventricle level pain test, the mice in the lateral ventricle should be operated in advance to ensure the accuracy of the drug injection site. The lateral ventricle of the mouse was buried using a brain stereotaxic instrument. Kunming male mice weighing 20±2 g; brain stereotaxic instrument is Jiangwan I. After the mice were anesthetized with sodium pentobarbital (ip, 80 mg/kg mouse body weight), the head operation area of the mouse was cut off and placed on the brain stereo positioner, and the operation area was disinfected with iodophor. The sagittal suture cuts the opening skin, reveals the skull, and finds the front squat position. From the front to the back 3 mm, to the left / right l mm, which is the upper position of the lateral ventricle. A self-made stainless steel tube (a section of 24-gauge, connected to a small section of PE-10 tube) is inserted down 3 mm in the above position, which is the lateral ventricle. A length of stainless steel string (28-gauge) is inserted into the steel tube to prevent cerebrospinal fluid from spilling, infecting and clogging the steel tube. The steel tube is fixed with medical powder and glue, and after solidification, the wound is sutured. After the operation, the mice recovered for 4 days, and the follow-up experiment was started on the 5th day. Surgical instruments, stainless steel tubes, etc. involved in the experiment have been disinfected before surgery. The drug is dissolved in physiological saline, stored at -20 ° C, and thawed before use.
( 2) 小鼠的鞘内和尾静脉注射 脊髓水平给药采用清醒小鼠鞘内注射法， 具体操作参考 Hylden和 Wilcox已报道的方法 ( Eur.丄 Pharmacol. 1980, 67:313)。将 25 μΙ微量进样器直接***到 L5和 L6之间的蛛网膜下腔。 蛛网膜被刺破时伴随着小鼠猛烈而明显的甩尾动作或者尾巴呈 "S' '形， 生理盐水和药物以 5 μΙ/10秒的速率注射至蛛网膜下腔中。 (2) Intrathecal and tail vein injections in mice Spinal cord administration is performed by intrathecal injection in conscious mice, and the specific procedure is described with reference to the methods reported by Hylden and Wilcox (Eur. Pharmacol. 1980, 67: 313). Insert the 25 μΙ microsampler directly into the subarachnoid space between L5 and L6. When the arachnoid was punctured, the mice had a violent and obvious appendix movement or the tail was "S'' shaped, and saline and drugs were injected into the subarachnoid space at a rate of 5 μΙ/10 sec.
尾静脉注射前用固定器将小鼠固定， 露出整个尾巴， 用 75%的酒精棉球擦拭注尾部射 位点使静脉扩张， 此时清晰可见尾部两侧红色的静脉。注射位点在靠近尾巴根部的 1/3-1/2 处。 选用带 4号半针头的 lml注射器， 注射体积为 0.1ml/只。 注射时用左手轻轻拉直固定尾 巴， 右手拿注射器沿着尾巴小于 30度角倾斜进针。 进针后， 30秒内完成药物注射， 注射完 成后用棉球压迫止血。  The mice were fixed with a fixator before the tail vein injection, exposing the entire tail, and the tail was sprayed with a 75% alcohol cotton ball to spread the veins, and the red veins on both sides of the tail were clearly visible. The injection site is located near 1/3-1/2 of the root of the tail. A lml syringe with a 4 gauge half needle was used with an injection volume of 0.1 ml/piece. Use a left hand to gently straighten the tail when injecting, and a syringe in the right hand to tilt the needle along the tail at an angle of less than 30 degrees. After the needle is inserted, the drug injection is completed within 30 seconds, and after the injection is completed, the cotton ball is pressed to stop bleeding.
( 3 ) 痛觉检测实验  (3) Pain detection test
使用昆明系雄性小鼠， 利用光热甩尾仪来检测药物对痛觉作用的影响。环境温度控制 在 22±1°C， 实验动物可自由进食、 饮水。 小鼠侧脑室每次注射 4 μΙ药物。 给药前先测定 基础甩尾潜伏期（3-5秒），过于敏感或迟钝的小鼠弃去不用。记录给药之后第 5， 10， 15， 20， 30， 40， 50, 60， 90分钟的甩尾潜伏期 （尾静脉注射时痛觉检测不测定 90分钟时间 点的甩尾潜伏期）。 为防止烫伤， 将 10秒设为最长甩尾潜伏期， 甩尾时间超过 10秒均以 10秒来计。  Kunming male mice were used to detect the effects of drugs on pain sensation using a photothermal appendix. The ambient temperature is controlled at 22±1 °C, and the experimental animals are free to eat and drink. The mouse lateral ventricle was injected with 4 μΙ of the drug each time. The basic appendix latency (3-5 seconds) was determined prior to dosing, and mice that were too sensitive or dull were discarded. The tail-flick latency of 5, 10, 15, 20, 30, 40, 50, 60, 90 minutes after administration was recorded (the pain detection at the tail vein injection did not measure the tail-flick latency at the 90-minute time point). To prevent burns, set the minimum tailing latency to 10 seconds and the tailing time to 10 seconds in 10 seconds.
( 4) 计算药物镇痛作用的 Μ ΡΕ和 EC5 (4) Μ and EC 5 for calculating the analgesic effect of drugs
痛觉调节作用利用最大可能效应 M PE ( maximum possible effect )值来评价， M PE ( ) = ΙΟΟχ [(给药后的痛阈一基础痛阈) /(10秒一基础痛阈)]。相关的 M PE数据用平均值 ±标准 误差 （Means±S.E.M. ) 表示， 不同药物浓度处理的组间差异用单因素方差分析 （one-way AN OVA的 Dunnett检验） 来进行数据的统计和分析， ***P < 0.001 表示药物处理组的 M PE 值与生理盐水组之间存在显著性差异。 实验结果见图 1~3。  The pain modulation effect was evaluated using the maximum possible effect MPE ( ) = ΙΟΟχ [(pain threshold after administration - basic pain threshold) / (10 seconds - basal pain threshold)]. The relevant M PE data were expressed as mean ± standard error (Means ± SEM ). Differences between groups treated with different drug concentrations were analyzed and analyzed by one-way analysis of variance (one-way AN OVA Dunnett test). **P < 0.001 indicates a significant difference between the M PE value of the drug-treated group and the saline group. The experimental results are shown in Figures 1~3.
BN-9和***的镇痛作用通过 EC5Q值进行比较。 (：5(5值表示药物产生 50%最大镇痛效应 时的药物剂量。利用统计学软件 GraphPad Prism 5.0版本， 分别绘制了 BN-9和***在侧脑 室、 鞘内和尾静脉三种不同给药水平的最大镇痛效应时间点的 M PE值的量效曲线 （见图 4~6 ) , 并计算出它们在不同注射水平的 EC5Q值和 95%的置信区间， 相关数据如表 1所示。 The analgesic effect of BN-9 and morphine was compared by EC 5Q values. (: 5 (5 values indicate the drug dose at which the drug produces a 50% maximal analgesic effect. Using the statistical software GraphPad Prism version 5.0, BN-9 and morphine were separately mapped in the lateral ventricle, intrathecal and tail vein. The dose-response curve of the M PE value at the maximum analgesic effect time point of the drug level (see Figures 4-6), and their EC 5Q values and 95% confidence intervals at different injection levels were calculated. The relevant data are shown in Table 1. Show.
表 1不同水平注射 BN-9和***所产生的镇痛作用的比较 a 、 , EC50值 （nmol) ( 95%的置信区间） EC50的比值 给药途径 9 « (*** /BN-9) 脑室 （i.c.v. ) 0.39 (0.33, 0.46) 1.02 (0.88, 1.18) .61倍 鞘内 （i.t. ) 0.29 (0.24, 0.34) 0.35 (0.30, 0.40) 1.21 倍 尾静脉 ( i.v. ) 58.6 (50.8, 67.4) 46.2 (38.7, 55.2) 0.79 倍 Table 1 Comparison of analgesic effects of different levels of BN-9 and morphine injection a , , EC 50 value (nmol) ( 95% confidence interval) EC 50 ratio administration route 9 « (morphine / BN-9) Ventricular (icv) 0.39 (0.33, 0.46) 1.02 (0.88, 1.18) .61 times Intrathecal (it) 0.29 (0.24, 0.34) 0.35 (0.30, 0.40) 1.21 times tail vein (iv) 58.6 (50.8, 67.4) 46.2 (38.7, 55.2) 0.79 times
生理盐水组为空白溶剂对照组， 侧脑室注射 BN-9的药物浓度分别为 0.25, 0.5, 1和 2 nmol。 每组动物数为 8只。 如图 1所示， 小鼠侧脑室注射新化合物 BN-9能剂量依赖地产 生镇痛作用。 BN-9在脊髓以上水平的镇痛作用能持续 60分钟， 并在药物注射后 15分钟 达到最大镇痛效应。 侧脑室注射*** （0.5, 1, 2和 4 nmol)， 其镇痛活性能持续 90分钟， 并在药物注射后 30分钟达到最大镇痛效应。 The saline group was a blank solvent control group, and the concentrations of BN-9 in the lateral ventricle were 0.25, 0.5, 1 and 2 nmol, respectively. The number of animals in each group was 8. As shown in Figure 1, the injection of the new compound BN-9 into the lateral ventricle of mice is dose-dependent and has an analgesic effect. The analgesic effect of BN-9 at the level above the spinal cord lasted for 60 minutes and reached the maximum analgesic effect 15 minutes after drug injection. Morphine (0.5, 1, 2, and 4 nmol) was injected into the lateral ventricle, and the analgesic activity lasted for 90 minutes, and the maximum analgesic effect was achieved 30 minutes after drug injection.
同样， 鞘内注射 BN-9 ( 0.125, 0.25, 0.5, 1禾 B 2 nmol ) 能引起量效依的镇痛作用， 该作 用在药物注射后 10分钟达到最强， 并能持续 60分钟 （如图 2所示）。 而脊髓水平注射的 ***（0.125, 0.25, 0.5, 1和 2 nmol ), 其镇痛活性只能持续 40分钟， 它在注射后 10分钟时 引起最大镇痛效应。  Similarly, intrathecal injection of BN-9 (0.125, 0.25, 0.5, 1 and B 2 nmol) can cause a dose-dependent analgesic effect, which is strongest at 10 minutes after drug injection and lasts for 60 minutes (eg Figure 2). The morphine (0.125, 0.25, 0.5, 1 and 2 nmol) injected horizontally on the spinal cord only lasted for 40 minutes, and it caused the greatest analgesic effect 10 minutes after the injection.
尾静脉注射 1.25, 2.5, 5和 10 mg的新嵌合肽 BN-9时能介导剂量依赖的镇痛活性， 其 最大镇痛效应时间点为药物注射后的 10分钟，并能维持镇痛作用 60分钟（如图 3所示）。 同样， 尾静脉注射的*** （0.25, 0.5, 1和 2 mg) 所引起的镇痛活性持续作用时间也为 60 分钟， 最大镇痛效应的时间点为药物注射后 10分钟。  Tail-injection of 1.25, 2.5, 5 and 10 mg of the new chimeric peptide BN-9 mediated dose-dependent analgesic activity with a maximum analgesic effect at 10 minutes after drug injection and maintenance of analgesia The effect is 60 minutes (as shown in Figure 3). Similarly, the analgesic activity caused by tail vein injection of morphine (0.25, 0.5, 1 and 2 mg) was also sustained for 60 minutes, and the maximum analgesic effect time was 10 minutes after drug injection.
如图 4~6和表 1所示， 比较 BN-9和***的镇痛量效曲线作用发现， 在侧脑室、 脊髓 和尾静脉三种不同的注射水平下， 新化合物 BN-9保持了较强的镇痛活性。 并且， 脊髓和 脊髓以上水平的 BN-9镇痛作用强于***的， 但尾静脉给药时， BN-9的镇痛作用稍弱于吗 啡的。  As shown in Figures 4-6 and Table 1, comparing the analgesic dose-effect curves of BN-9 and morphine, the new compound BN-9 was maintained at three different injection levels in the lateral ventricle, spinal cord and tail vein. Strong analgesic activity. Moreover, the analgesic effect of BN-9 at the level of spinal cord and above the spinal cord was stronger than that of morphine, but the analgesic effect of BN-9 was slightly weaker than that of morphine when administered intravenously.
2、 BN-9的镇痛耐受实验  2. Analgesic tolerance test of BN-9
小鼠侧脑室连续八天注射药物， 利用光热甩尾法检测它们的镇痛耐受现象， 进一步比 较本发明的化合物 BN-9和***在镇痛耐受方面的药理学活性。  The mice were injected with the drug for eight consecutive days in the lateral ventricle of the mice, and their analgesic tolerance was examined by photothermal appendix method, and the pharmacological activity of the compound of the present invention BN-9 and morphine in analgesic tolerance was further compared.
( 1) 小鼠的痛觉耐受实验方法  (1) Experimental method for pain tolerance in mice
昆明系雄性小鼠， 侧脑室或鞘内连续注射药物八天， 每天注射一次， 利用光热甩尾仪 来检测药物连续注射对痛觉作用的影响。环境温度控制在 22±1°C，实验动物可自由进食、 饮水。 小鼠侧脑室每次注射 4 μΙ药物。 第一天药物注射前测定基础甩尾潜伏期 （3-5秒）， 过于敏感或迟钝的小鼠弃去不用。实验中记录给药之后相关药物最大镇痛效应时间点的甩 尾潜伏期， 如侧脑室注射 ΒΝ-9和***的记录时间点分别为给药后 15和 30分钟； 鞘内注 射这两个药物的记录时间点均为给药物 10分钟；空白溶剂对照组的时间记录点与 ΒΝ-9的 保持一致。 为防止小鼠尾巴烫伤， 将 10秒设为最长甩尾潜伏期。 Male Kunming mice were injected intracerebroventricularly or intrathecally for eight days, once a day, using a photothermal appendix to detect the effect of continuous drug injection on pain perception. The ambient temperature is controlled at 22 ± 1 ° C, and the experimental animals are free to eat and drink. The mouse lateral ventricle was injected with 4 μΙ of the drug each time. The baseline tail-flick latency (3-5 seconds) was measured before the first day of drug injection, and mice that were too sensitive or dull were discarded. The tail time latency of the maximum analgesic effect time of the relevant drug after administration was recorded in the experiment, such as the recording time points of the lateral ventricle injection of ΒΝ-9 and morphine were 15 and 30 minutes after administration, respectively; intrathecal injection of these two drugs Recording time points were given to the drug for 10 minutes; the time of the blank solvent control group was recorded with ΒΝ-9 be consistent. To prevent burns on the tail of the mouse, set the maximum tailing latency to 10 seconds.
( 2) 耐受实验数据统计  (2) Tolerance experiment data statistics
实验数据用 MPE ( maximum possible effect) 表示， MPE ( ) = 100χ[ (给药后的痛 阈一基础痛阈） I ( 10秒一基础痛阈） ]。 药物的镇痛耐受作用利用相关药物最大镇痛效应 时间点的 ΜΡΕ值来进行比较。 ΜΡΕ数据用平均值 ±标准误差 （Means±S.E.M. ) 来表示， 小 鼠八天镇痛作用的差异用单因素方差分析（one-way AN0VA的 Bonferroni检验)进行统计， ***P < 0.001表示与第一天***镇痛作用相比有显著性差异。 实验结果如图 7、 8所示。  The experimental data is expressed by MPE (maximum possible effect), MPE ( ) = 100 χ [(pain threshold after administration - basic pain threshold) I (10 seconds - basic pain threshold)]. The analgesic tolerance of the drug was compared using the maximum analgesic effect of the drug at the time point of the sputum. ΜΡΕ Data were expressed as mean ± standard error (Means ± SEM). Differences in eight-day analgesic effects in mice were statistically analyzed by one-way ANOVA (one-way AN0VA Bonferroni test), ***P < 0.001 indicates There was a significant difference in the analgesic effect of morphine on the first day. The experimental results are shown in Figures 7 and 8.
生理盐水组为空白溶剂对照组， 侧脑室注射 BN-9的药物剂量为 0.5, 1, 2和 4 nmol, ***的剂量为 4 nmol o图 7的实验数据表明小鼠侧脑室连续八天注射空白溶剂对照生理盐 水后， 小鼠都不产生镇痛活性。 与空白溶剂对照生理盐水组相比， 第一天侧脑室注射*** 和各种剂量的 BN-9都显著地增加了小鼠的甩尾潜伏期。 但小鼠侧脑室连续注射八天*** 组， 在给药后第四天 MPE值开始显著地下降， 并持续至第八天， 即***镇痛从第四天起 就开始出现了镇痛耐受现象。与第一天相比， 第四天至第八天的差异性均为极其显著（P < 0.001)。 然而， 0.5, 1, 2和 4 nmol的 BN-9侧脑室连续注射期间， 其镇痛作用的 MPE值都 无明显变化 （P > 0.05)， 在连续注射的八天内一直保持较好的无耐受镇痛活性。  The saline group was a blank solvent control group, the dose of BN-9 in the lateral ventricle was 0.5, 1, 2 and 4 nmol, and the dose of morphine was 4 nmol. The experimental data in Figure 7 showed that the lateral ventricle of the mice was injected for eight consecutive days. After the solvent was compared with physiological saline, the mice did not produce analgesic activity. Compared with the blank solvent control saline group, the intraventricular injection of morphine and various doses of BN-9 on the first day significantly increased the tail-flick latency of the mice. However, in the lateral ventricle of mice, the morphine group was continuously injected for eight days. On the fourth day after the administration, the MPE value began to decrease significantly and continued until the eighth day, that is, morphine analgesia began to appear analgesic tolerance from the fourth day. phenomenon. The difference between the fourth day and the eighth day was extremely significant compared with the first day (P < 0.001). However, during the continuous injection of BN-9 lateral ventricle at 0.5, 1, 2 and 4 nmol, there was no significant change in the MPE value of analgesic effect (P > 0.05), and it remained well tolerant within eight days of continuous injection. It is analgesic.
与脊髓以上水平的镇痛耐受检测结果相一致， 在脊髓水平连续八天注射 0.5, 1 和 2 nmol的 BN-9, 均无镇痛耐受现象出现。 与第一天的 MPE相比， 不同剂量 BN-9在第二天 至第八天的相应镇痛效应均无明显的变化。 但是， 鞘内注射 2 nmol的***， 在连续给药 后的第 4天就开始出现镇痛耐受， 并且其镇痛耐受逐渐加强。 具体结果如图 8所示。  Consistent with the results of the analgesic tolerance test at the level above the spinal cord, there was no analgesia tolerance in the 0.5, 1 and 2 nmol BN-9 injected for eight consecutive days at the spinal cord level. There was no significant change in the corresponding analgesic effects of different doses of BN-9 between the second and eighth days compared with the first day of MPE. However, intrathecal injection of 2 nmol of morphine began to develop analgesia on the fourth day after continuous administration, and its analgesic tolerance gradually increased. The specific results are shown in Figure 8.
3、 BN-9胃肠运动调节作用的检测  3. Detection of BN-9 gastrointestinal motility regulation
阿片类镇痛药物除了耐受外， 还能引起便秘等副作用。 因此， 对嵌合肽 BN-9和*** 在胃肠运动调节方面的作用作了进一步的评价。通过小鼠侧脑室注射药物， 用碳粉胃肠运 动检测法评价了它们对在体胃肠运动的影响。  In addition to tolerance, opioid analgesics can cause side effects such as constipation. Therefore, the role of the chimeric peptide BN-9 and morphine in the regulation of gastrointestinal motility was further evaluated. Drugs were injected into the lateral ventricles of mice, and their effects on gastrointestinal motility were evaluated by the toner gastrointestinal motility assay.
( 1) 小鼠的在体胃肠运动检测方法  (1) In vivo gastrointestinal motility detection method in mice
昆明系雄性小鼠， 体重 20±2 g。 侧脑室埋管后恢复 4天后用于胃肠运动检测。 在体胃 肠运动检测选用常用的碳粉检测法 ( Peptides, 2000, 21:295 )。 具体的实验过程为： 胃肠运 动实验前小鼠禁食 16小时 （禁食期间可随意饮水）， 然后侧脑室注射药物， 15分钟后将 预先准备好的活性炭悬浮液 （一种含 5%活性炭和 10%***树胶的生理盐水悬浮液） 以 每 10 克体重 0.1 ml的体积经口灌注到胃。 活性炭悬浮液灌注 30分钟后， 颈部脱白处死 动物， 然后仔细地取出从幽门到盲肠的动物小肠。测量幽门到盲肠的长度以及活性炭悬浮 液移动的最远距离。 Kunming male mice, weighing 20 ± 2 g. The lateral ventricle was restored to the gastrointestinal motion test after 4 days of recovery. The commonly used toner detection method is used for the detection of body gastrointestinal motility (Peptides, 2000, 21:295). The specific experimental procedure is as follows: Before the gastrointestinal exercise experiment, the mice were fasted for 16 hours (free drinking during fasting), then the side ventricle was injected with drugs, and the activated carbon suspension prepared in advance after 15 minutes (a 5% activated carbon) And a physiological saline suspension of 10% gum arabic) was orally infused into the stomach in a volume of 0.1 ml per 10 g of body weight. After the activated carbon suspension was perfused for 30 minutes, the neck was defeated and the animals were sacrificed, and then the small intestine of the animal from the pylorus to the cecum was carefully removed. Measuring the length of the pylorus to the cecum and the suspension of activated carbon The farthest distance the liquid moves.
( 2) 在体胃肠运动实验数据统计  (2) Statistical data of body gastrointestinal exercise experiment
胃肠运动实验结果用胃肠运动百分比来评价，每只小鼠的胃肠运动百分比通过活性炭 悬浮液移动距离除以小肠总长度之后的百分比来计算。相关的数据用胃肠运动百分比的平 均值 ±标准误差 （MeanS±S.E.M. ) 来表示， 不同药物浓度处理的组间差异用单因素方差分 析 （one-way AN OVA的 Bonferroni检验） 进行数据统计和分析， < 0.01, ***P < 0.001 表 示药物处理组的胃肠运动百分比与生理盐水组的之间存在显著性差异。实验结果如图 9所 示。 Gastrointestinal exercise test results were evaluated as a percentage of gastrointestinal motility, and the percentage of gastrointestinal motility per mouse was calculated by dividing the moving distance of the activated carbon suspension by the percentage after the total length of the small intestine. The relevant data were expressed as mean ± standard error (Mea nS ± SEM) of the percentage of gastrointestinal motility, and differences between groups treated with different drug concentrations were statistically analyzed by one-way analysis of variance (one-way AN OVA Bonferroni test). Analysis, < 0.01, ***P < 0.001 indicates a significant difference between the percentage of gastrointestinal motility in the drug-treated group and the saline group. The experimental results are shown in Figure 9.
在图 9中， 生理盐水组为空白溶剂对照组， ***的药物浓度为 4 nmol， BN-9的药物 浓度分别为 0.5, l, 2和4 nmOl。 每组小鼠数为 9只。 实验结果表明， 空白溶剂对照组的胃 肠运动百分比为 80 %， 与生理盐水组相比， 侧脑室注射 4 nmol的***组显著地抑制小鼠 胃肠运动， 其胃肠运动百分比仅为 14%。 然而， 小鼠侧脑室注射低剂量的 BN-9 ( 0.5禾口 1 nmol ), 对小鼠的胃肠运动几乎无影响； 当注射 2和 4 nmol的 BN-9时， 胃肠运动百分比 分别为 58%和 34%， 高剂量的 BN-9能明显地抑制小鼠的胃肠运动， 但抑制作用强度低于 ***的。 由于 BN-9在侧脑室水平镇痛效应的 (：5(5值要低于***， 因此， 在有效镇痛剂量 范围内， BN-9的便秘副作用要远远低于***。 In Fig. 9, the saline group was a blank solvent control group, the morphine concentration was 4 nmol, and the BN-9 drug concentrations were 0.5, 1, 2, and 4 n m O l , respectively. The number of mice in each group was 9. The experimental results showed that the percentage of gastrointestinal motility in the blank solvent control group was 80%. Compared with the saline group, the morphine group injected into the lateral ventricle 4 nmol significantly inhibited gastrointestinal motility in mice, and the percentage of gastrointestinal motility was only 14%. . However, the injection of low doses of BN-9 (0.5 and 1 nmol) into the lateral ventricle of mice had little effect on gastrointestinal motility in mice; when 2 and 4 nmol of BN-9 were injected, the percentage of gastrointestinal motility was At 58% and 34%, high doses of BN-9 significantly inhibited gastrointestinal motility in mice, but the inhibitory effect was lower than that of morphine. Because of the level of analgesic effect of BN-9 in the lateral ventricle ( 5 (5 values are lower than morphine), the constipation side effects of BN-9 are much lower than morphine in the effective analgesic dose range.
4、 中枢注射 BN-9对***致痛作用的调节  4, central injection BN-9 regulation of formalin-induced pain
为了进一步评价 BN-9的镇痛作用， 利用临床前镇痛药物评价方法研究了嵌合肽 BN-9 对炎症痛的调节作用。在小鼠***致痛模型中，分别评价了 BN-9和***的镇痛活性。  To further evaluate the analgesic effect of BN-9, the regulatory effect of chimeric peptide BN-9 on inflammatory pain was studied using preclinical analgesic drug evaluation methods. The analgesic activity of BN-9 and morphine was evaluated in a mouse formalin-induced pain model.
( 1) 小鼠***致痛实验方法  (1) Experimental method of mouse formalin-induced pain
昆明系雄性小鼠， 体重 20±2 g。 侧脑室埋管后第 5天用于***致痛实验。 环境温 度控制在 22±1°C， 实验动物可自由进食、 饮水。 具体的检测方法参考」 ohn Wiley & Sons 出版社 (John Wiley & Sons, Inc. ) 2007出版的 Curr. Protoc. Neurosci. ( 8.9.1-8.9.16. ) 一书。 具体实验操作为：将小鼠放入有机玻璃盒中（20x20x30 cm ),先使其适应环境 10-15分钟， 然后给小鼠侧脑室注射药物， 给药体积为 5 μΙ。 给药 5 min后， 将小鼠取出， 用手轻柔的 抓住小鼠颈部及后背的皮肤， 小拇指压住小鼠的尾巴， 腹部朝上固定小鼠且露出小鼠的后 爪。 在小鼠的右后脚掌中间皮肤下注射 20 μΙ的***溶液 （浓度为 5 %)， 迅速将小鼠 放回观察盒中， 同时开始计时， 并记录注射***溶液后 40 分钟内小鼠行为学反应。 ***一般能引起两个时相的反应， 根据实验需要， 采用了两种行为学记录方法： （1) 记录注射***后每隔 1分钟小鼠舔、 咬、 抖被注射足的时间， 共记录 40 分钟； （2) 记录注射***后 0~5 分钟 （第一相）和 15-30分钟 （第二相） 内小鼠舔、 咬、 抖被注 射足的时间。 Kunming male mice, weighing 20 ± 2 g. On the 5th day after the lateral ventricle was buried, it was used for the formalin-induced pain test. The ambient temperature is controlled at 22 ± 1 ° C, and the experimental animals are free to eat and drink. Refer to Curr. Protoc. Neurosci. ( 8.9.1-8.9.16.), published by John Wiley & Sons, Inc. 2007, for specific test methods. The specific experimental procedure was as follows: the mice were placed in a plexiglass box (20 x 20 x 30 cm), first adapted to the environment for 10-15 minutes, and then the mice were injected with the drug into the lateral ventricle at a dose of 5 μΙ. After 5 minutes of administration, the mice were taken out, and the skin of the neck and back of the mouse was gently grasped by hand, the little finger was pressed against the tail of the mouse, and the mouse was fixed with the abdomen facing upward to expose the hind paw of the mouse. A 20 μM formalin solution (5 % concentration) was injected subcutaneously into the skin of the right hind paw of the mouse, and the mice were quickly returned to the observation box, and timing was started, and 40 minutes after the injection of formalin solution was recorded. Mouse behavioral response within minutes. Formalin generally causes two phases of response. According to the experimental needs, two behavioral recording methods were used: (1) The mice were injected, bitten, and shaken every 1 minute after injection of formalin. A total of 40 minutes of recording time; (2) The time to inject the foot in the mouse, bite, and shake was recorded within 0 to 5 minutes (first phase) and 15-30 minutes (second phase) after injection of formalin.
( 2) 小鼠***致痛实验结果的统计  (2) Statistics on the results of mouse formalin-induced pain test
根据两种不同的行为学记录方法，药物对***致痛作用的影响结果统计方法也分 为两种： （1)根据每隔 1分钟所记录的舔、 咬、 抖被注射足的时间， 来评价不同药物对福 尔马林致痛作用的反应， 具体见图 10; ( 2)药物对***痛的调节利用 MPE ( maximum possible effect) 值来评价， MPE ( ) = 100χ [ (空白溶剂对照组的舔、 咬、 抖被注射足时 间一药物组的舔、 咬、 抖被注射足时间) /( 空白溶剂对照组的舔、 咬、 抖被注射足时间)]。 数据用平均值 ±标准误差（MeanS±S.E.M. )表示，不同药物处理组各时间段内所测定的 MPE 值与空白溶剂对照组之间的差异用单因素方差 （ one-way AN0VA的 Bonferroni检验） 来进 行分析和统计， **P < 0.01, ***P < 0.001 表示药物处理组的 MPE值与生理盐水组相同时间段 内的 MPE值相比差异显著。 实验结果如图 11和图 12所示。 According to two different behavioral recording methods, the statistical methods for the effects of drugs on formalin-induced pain are also divided into two types: (1) according to the sputum, bite, and shaking recorded every 1 minute. Time, to evaluate the response of different drugs to formalin-induced pain, as shown in Figure 10; (2) The regulation of formalin pain by the drug is evaluated by MPE (maximum possible effect) value, MPE ( ) = 100χ [(Blank solvent control group, sputum, bite, and shaking were injected at a time when a drug group was licked, bitten, and shaken were injected for a full time) / (the blank solvent control group was licked, bitten, and shaken were injected for a full time)]. Data were expressed as mean ± standard error (Mea nS ± SEM). Differences between the MPE values measured in the different drug treatment groups and the blank solvent control group were analyzed by one-way variance (one-way AN0VA Bonferroni test). For analysis and statistics, **P < 0.01, ***P < 0.001 indicates that the MPE value of the drug-treated group was significantly different from the MPE value in the same time period of the saline group. The experimental results are shown in Figures 11 and 12.
图 10中， 生理盐水组为空白溶剂对照组， BN-9和***处理组的药物浓度分别为 5和 lO nmoL 每组动物数为 7~10只。 实验结果表明小鼠侧脑室注射生理盐水后， ***会 引起两个时相的痛觉反应， 即 0~5 分钟的第一相的急痛反应和 15~30 分钟的第二相炎症 痛反应。 而侧脑室注射 5 nmol的 BN-9和 10 nmol的***能明显地抑制***所引起两 相痛觉反应。 进一步研究了 BN-9和***分别对第一相和第二相反应的镇痛作用的量效关 系，具体结果如图 11和图 12所示。侧脑室注射 BN-9的药物剂量为 0.625, 1.25, 2.5禾 B 5 nmol, ***的剂量为 1.25, 2.5, 5和 10 nmol。实验结果表明不同剂量的 BN-9和***都对第一相急 性痛具有较强的镇痛活性， 其抑制率均在 70 ~ 100%之间。 而且， BN-9和***能剂量依赖 地抑制***所引起的第二相炎症痛， 其量效曲线显示， BN-9 对第二相炎症痛的镇痛 作用稍强于***。  In Fig. 10, the saline group was a blank solvent control group, and the BN-9 and morphine treatment groups had a drug concentration of 5 and 10 nmoL, respectively, and the number of animals per group was 7 to 10. The experimental results showed that formalin caused two pain sensations after the injection of normal saline into the lateral ventricle, ie, the acute phase reaction of the first phase from 0 to 5 minutes and the second phase inflammation pain of 15 to 30 minutes. reaction. Injection of 5 nmol of BN-9 and 10 nmol of morphine into the lateral ventricle significantly inhibited the two-phase pain response induced by formalin. The dose-effect relationship between BN-9 and morphine for the analgesic effects of the first phase and the second phase, respectively, was further studied. The specific results are shown in Fig. 11 and Fig. 12. The dose of BN-9 injected into the lateral ventricle was 0.625, 1.25, 2.5 and B 5 nmol, and the doses of morphine were 1.25, 2.5, 5 and 10 nmol. The experimental results show that different doses of BN-9 and morphine have strong analgesic activity on the first phase of acute pain, and the inhibition rate is between 70 and 100%. Moreover, BN-9 and morphine dose-dependently inhibited the second phase of inflammatory pain caused by formalin. The dose-response curve showed that BN-9 had a slightly stronger analgesic effect on the second phase of inflammatory pain than morphine.
综上所述， 本发明基于阿片肽 Biphalin和 NPFF的嵌合肽 BN-9表现出比***更强的中 枢镇痛活性， 并具有无镇痛耐受、 对胃肠运动影响小等优点， 有效克服了阿片类镇痛药物 普遍存在的耐受和便秘等副作用问题。 此外， 侧脑室注射 BN-9能显著地抑制***所 引起的急性痛和炎症痛， 且镇痛作用稍强于***， 因此该嵌合肽 BN-9具有治疗临床疼痛 的潜在应用价值。  In summary, the chimeric peptide BN-9 based on the opioid peptide Biphalin and NPFF exhibits stronger central analgesic activity than morphine, and has the advantages of no analgesic tolerance and little effect on gastrointestinal motility, and is effective. Overcoming side effects such as tolerance and constipation, which are common in opioid analgesics. In addition, intracerebroventricular injection of BN-9 significantly inhibited the acute pain and inflammatory pain caused by formalin, and the analgesic effect was slightly stronger than morphine, so the chimeric peptide BN-9 has potential application value in the treatment of clinical pain. .
附图说明 DRAWINGS
图 1为小鼠侧脑室注射 BN-9所产生的剂量依赖性镇痛作用的时效曲线；  Figure 1 is a time-effect curve of dose-dependent analgesia produced by injection of BN-9 into the lateral ventricle of mice;
图 2为小鼠鞘内注射 BN-9所产生的剂量依赖性镇痛作用的时效曲线； 图 3为小鼠尾静脉注射 BN-9所产生的剂量依赖性镇痛作用的时效曲线。（A)为注射； ( B ) 为注射； Figure 2 is a time-effect curve of dose-dependent analgesic effect produced by intrathecal injection of BN-9 in mice; Figure 3 is a time-effect curve of dose-dependent analgesic effects produced by tail vein injection of BN-9 in mice. (A) for injection; (B) for injection;
图 4为小鼠侧脑室注射 BN-9和***所产生的镇痛作用的量效曲线；  Figure 4 is a dose-response curve of the analgesic effect of BN-9 and morphine injected into the lateral ventricle of mice;
图 5为小鼠鞘内注射 BN-9和***所产生的镇痛作用的量效曲线；  Figure 5 is a dose-response curve of the analgesic effect of intrathecal injection of BN-9 and morphine in mice;
图 6为小鼠尾静脉注射 BN-9和***所产生的镇痛作用的量效曲线；  Figure 6 is a dose-response curve of the analgesic effect of BN-9 and morphine injected into the tail vein of mice;
图 7为小鼠脊髓和脊髓以上水平连续八天侧脑室注射 BN-9和***所引起的镇痛作用 的变化；  Figure 7 shows the changes in analgesic effects caused by intracerebroventricular injection of BN-9 and morphine for eight consecutive days in the spinal cord and spinal cord of mice;
图 8为小鼠脊髓和脊髓以上水平连续八天鞘内注射 BN-9和***所引起的镇痛作用的 变化；  Figure 8 shows the changes in analgesic effects caused by intrathecal injection of BN-9 and morphine for eight consecutive days in the spinal cord and spinal cord of mice;
图 9 小鼠侧脑室注射 BN-9和***分别对胃肠运动的影响；  Figure 9 Effect of BN-9 and morphine on gastrointestinal motility in the lateral ventricle of mice;
图 10小鼠脑室注射高剂量 BN-9和***分别***所引起的急性痛和炎症痛的抑制 作用；  Figure 10 Inhibition of acute pain and inflammatory pain caused by high doses of BN-9 and morphine in formalin in mice;
图 11小鼠脑室注射 BN-9和***分别***所引起的急性痛（第一相）抑制的量效 曲线；  Figure 11 is a dose-effect curve of acute pain (first phase) inhibition by formalin injection of BN-9 and morphine in formalin;
图 12小鼠脑室注射 BN-9和***分别***所引起的炎症痛（第二相）抑制的量效 曲线。  Figure 12 is a dose-effect curve of inflammatory pain (second phase) inhibition by formalin injection of BN-9 and morphine, respectively.
具体实施方式 detailed description
下面通过具体实施例对本发明的嵌合肽合成方法作进一步说明。  The chimeric peptide synthesis method of the present invention will be further illustrated by the following specific examples.
I 材料  I material
仪器： 高效液相色谱仪 （HPLC ) 为 Waters公司的 Delta 600; 分析柱： DELTA PAK 5μ〔18 300A 3.9xl50mm ;制备柱： DELTA PAK 15μ C18 300 Λ 7.8x300mm。质谱仪为 PE Biosystems, Mariner System 5074。 手动固相多肽合成仪， 由本实验室设计后由玻璃工制作 （合成仪的 设计原理具体参考 Chen WC和 White PD所编的 《Fmoc solid phase peptide synthesis)) 中第 14 页图 4， 并在其基础上作了部分改进， 即用以机械搅拌方式替代鼓氮气法， 从而达到反应 溶液充分混合的目的）。 试齐 U: 树脂为 Rink-Amide-M BHA-Resin ( 1% DVB, 200 ~ 400 mesh , 取代值 S = 0.40 mmol/g resin ) ,购自天津南开和成公司。 A/-a-Fmoc保护的氨基酸（Fmoc-Aa )、 Λ/-羟基苯并三氮唑（HOBt)、 0-苯并三氮唑 -Λ/,Λ/,Λ/',Λ/ '-四甲基脲 -六氟磷酸盐（HBTU )、 二异 丙基乙胺 （DI EA)和三异丙基硅烷（TIS )购自吉尔生化（上海）有限公司。 茚三酮为上海 试剂三厂产品。 二氯甲烷 （DCM )、 Λ/,Λ/-二甲基甲酰胺 （DMF)、 六氢吡啶 （哌啶）、 甲醇 ( MeOH ) 和吡啶都购自天津第二试剂厂， 三氟乙酸 （TFA)、 苯酚和吡啶均为天津试剂一 厂产品； 以上有机试剂使用前均经过重蒸处理。 Apparatus: High performance liquid chromatography (HPLC) was Waters' Delta 600; analytical column: DELTA PAK 5μ [18 300A 3.9xl50mm ; preparative column: DELTA PAK 15μ C18 300 Λ 7.8x300mm. The mass spectrometer was PE Biosystems, Mariner System 5074. The manual solid phase peptide synthesizer was designed by the laboratory and made by the glassworker (the design principle of the synthesizer is specifically referred to the "Fmoc solid phase peptide synthesis" compiled by Chen WC and White PD), and is shown in Fig. 4 on page 14 On the basis of the partial improvement, the mechanical stirring method is used instead of the drum nitrogen method to achieve the purpose of thorough mixing of the reaction solution. Try U: Resin for Rink-Amide-M BHA-Resin (1% DVB, 200 ~ 400 mesh, substituted value S = 0.40 mmol / g resin), purchased from Tianjin Nankai Hecheng Company. A/-a-Fmoc protected amino acid (Fmoc-Aa), Λ/-hydroxybenzotriazole (HOBt), 0-benzotriazole-Λ/, Λ/, Λ/', Λ/ '- Tetramethylurea-hexafluorophosphate (HBTU), diisopropylethylamine (DI EA) and triisopropylsilane (TIS) were purchased from Jill Biochemical (Shanghai) Co., Ltd. Ninhydrin is a product of Shanghai Reagent III. Dichloromethane (DCM), hydrazine/, hydrazine/-dimethylformamide (DMF), hexahydropyridine (piperidine), methanol (MeOH) and pyridine were purchased from Tianjin Second Reagent Factory, trifluoroacetic acid (TFA). ), phenol and pyridine are all Tianjin reagents Factory products; The above organic reagents are re-steamed before use.
II 粗肽合成  II crude peptide synthesis
采用 Fmoc保护策略的固相多肽合成法。 肽链延长采用逐一接肽法 （step by SteP)。 具 体操作步骤如下： Solid phase peptide synthesis using Fmoc protection strategy. Peptide chain elongation was performed by peptide method (st ep by S te P ). The specific steps are as follows:
( 1)树脂预处理：将 500 mg Rink-Amide-MBHA树脂加入合成仪中，再加入 8 ml的 DCM 后搅拌 30 min， 使树脂充分溶胀后减压抽干溶剂。  (1) Resin pretreatment: 500 mg of Rink-Amide-MBHA resin was added to the synthesizer, and 8 ml of DCM was added and stirred for 30 min to fully swell the resin and then drain the solvent under reduced pressure.
( 2)脱除 Fmoc基团保护： 在溶胀、抽干溶剂的树脂中， 加入 8 ml体积浓度 20 %的六 氢吡啶 /DMF溶液， 搅拌 5 min后抽干， 重复 2次。再加入 8 ml 的体积浓度 20 %的六氢吡 啶 /DM F溶液， 搅拌 15 min 后抽干。 最后力口入 8 ml 的 DMF， 揽摔 3 min后抽干， 重复 4 次， 得到脱除 Fmoc基团保护的树脂样品。  (2) Removal of Fmoc group protection: In a resin which swells and drains the solvent, 8 ml of a 20% hexahydropyridine/DMF solution is added, stirred for 5 minutes, and then drained and repeated twice. Add 8 ml of 20% hexahydropyridine/DM F solution, stir for 15 min, and drain. Finally, 8 ml of DMF was injected into the mouth, and the mixture was drained for 3 minutes, then drained and repeated 4 times to obtain a resin sample from which the Fmoc group was removed.
( 3 ) 茚检： 按以下顺序加入试管中： 树脂样品、 0.1 ml试剂①、 0.2ml试剂②、 0.1 ml 试剂③， 沸水浴 3-10 min后观察。 溶液、 树脂均为蓝色表示 Fmoc保护基团脱除完全。 用 于茚检的三种试剂的配方为： 试剂① 80 g苯酚 /20ml乙醇； 试剂② 2.0 ml的 0.001 M氰化 钾 （水） / 98ml吡啶； 试剂③ 5 g 茚三酮 /100 ml乙醇。  (3) Inspection: Add the test tube in the following order: Resin sample, 0.1 ml reagent 1, 0.2 ml reagent 2, 0.1 ml reagent 3, and observe in a boiling water bath for 3-10 min. The blue solution and the resin indicate that the Fmoc protecting group is completely removed. The three reagents used for the sputum test were: reagent 1 80 g phenol / 20 ml ethanol; reagent 2 2.0 ml of 0.001 M potassium cyanide (water) / 98 ml pyridine; reagent 3 5 g ninhydrin / 100 ml ethanol.
(4)縮合： 在小烧杯中用 DMF依次将 A/-a-Fmoc保护基团氨基酸（Fmoc-Aa )、 Λ/-羟基 苯并三氮唑（HOBt)、 0-苯并三氮唑 -Λ/,Λ/,Λ/',Λ/ '-四甲基脲 -六氟磷酸盐（HBTU ) 以 1:1:1的摩 尔比完全溶解， 再加入 Fmoc-Aa两倍量的二异丙基乙胺 （DIEA) 后充分混匀， 得混合溶 液； 将 8 ml混合溶液和步骤 （2) 得到的脱除 Fmoc基团保护的树脂加入到合成仪中， 在 氩气保护下搅拌反应 60 min， 抽干溶剂。 加入 8 ml 的 DMF， 搅拌 3 min后抽干， 重复 3 次， 得肽树脂样品； 按照步骤 （3 ) 进行茚检， 茚检溶液为淡黄、 树脂为无色表示縮合完 全。  (4) Condensation: A/-a-Fmoc protecting group amino acid (Fmoc-Aa), Λ/-hydroxybenzotriazole (HOBt), 0-benzotriazole- in sequence in a small beaker. Λ/,Λ/,Λ/',Λ/ '-tetramethylurea-hexafluorophosphate (HBTU) is completely dissolved in a molar ratio of 1:1:1, and then twice the amount of diisopropyl of Fmoc-Aa After mixing with ethylamine (DIEA), the solution is mixed; 8 ml of the mixed solution and the Fmoc group-protected resin obtained in the step (2) are added to the synthesizer, and the reaction is stirred under argon for 60 min. , drain the solvent. Add 8 ml of DMF, stir for 3 min, drain dry, and repeat 3 times to obtain a peptide resin sample; follow the step (3) for sputum examination, the sputum test solution is light yellow, and the resin is colorless to indicate complete condensation.
( 5 )肽链的延长： 重复步骤 （2)、 （4) 八次， 按照嵌合肽结构由 C-末端向 N-末端的顺 序， 依次将带有 /V-a-Fmoc保护基团氨基酸逐个縮合， 每步反应完成后， 经负压作用过滤 去除反应器中试剂， 直至完成所有氨基酸残基的縮合。  (5) Extension of the peptide chain: repeat steps (2), (4) eight times, according to the chimeric peptide structure from the C-terminus to the N-terminus, sequentially condense the amino acids with /Va-Fmoc protecting group one by one. After each reaction is completed, the reagent in the reactor is removed by vacuum filtration until the condensation of all amino acid residues is completed.
III 肽链从树脂上的切割  III peptide chain cutting from resin
肽链的氨基酸残基全部縮合完成后， 按照上述步骤 （2) 中操作将肽树脂最后一个氨 基酸的 Fmoc基团脱除完全。然后按照 DCM 3 minx2次， MeOH 3 minxl次； DCM 3 minxl次， MeOH 3 minx2次的操作交替洗树脂。 移开搅棒， 将合成仪密封（胶塞）， 彻底抽干（至少 2 小时）。 将干燥的肽链树脂置于反应器中， 加入 18ml的切割剂 （体积比为 TFA:TIS^ = 95:2.5:2.5 ), 于室温下切割反应 2.5小时 （每 15分钟搅拌一次， 每次搅拌 1分钟）。 过滤， 滤 液在不高于 37°C的条件下充分减压旋干， 然后用不高于 -10°C的***析出沉淀， 振荡使粗 肽以白色沉淀的形式充分析出。 静置后将上清液吸出， 加水充分溶解析出的沉淀， 用分液 漏斗将***从水相中分离除去， 合并水相通过冷冻干燥得到白色的粗肽固体粉末 127.8 mg, 产率为 57.5 %。 After all the amino acid residues of the peptide chain have been completely condensed, the Fmoc group of the last amino acid of the peptide resin is completely removed according to the procedure in the above step (2). Then, according to DCM 3 min x 2 times, MeOH 3 min x 1 time; DCM 3 min x 1 times, MeOH 3 min x 2 times alternately wash the resin. Remove the stir bar, seal the synthesizer (gel plug), and drain thoroughly (at least 2 hours). The dried peptide chain resin was placed in a reactor, 18 ml of a cutting agent (volume ratio TFA: TIS^ = 95:2.5:2.5) was added, and the reaction was cut at room temperature for 2.5 hours (stirred every 15 minutes, each stirring) 1 minute). Filter, filter The solution was sufficiently dried under reduced pressure at a temperature not higher than 37 ° C, and then precipitated with diethyl ether not higher than -10 ° C, and shaken to allow the crude peptide to be analyzed as a white precipitate. After standing, the supernatant was aspirated, water was added to dissolve the precipitate, and the ether was separated from the aqueous phase by a separating funnel. The combined aqueous phases were lyophilized to give a white crude solid powder 127.8 mg, yield 57.5 %. .
IV粗肽的脱盐和纯化。  Desalting and purification of IV crude peptide.
将全部粗产物溶于 20%乙酸溶液中， 将溶液过 Sephadex G25交联葡聚糖凝胶柱 ( 2.0x25cm ) , 流动相为体积浓度 20%乙酸溶液。 利用紫外检测仪收集主峰后冷冻干燥， 得到脱盐处理后的白色粉末 116.2 mg。 再用反向高效液相色谱 （HPLC) C18柱 （XBridge TM BEH 130 C18， 19 mmx250mm ) 对上述脱盐的肽化合物进行分离纯化， 经分离后收集样品 主峰， 冷冻干燥得到白色的纯肽固体粉末 82.2 mg o 纯化后的 BN-9样品纯度在 98 %以上， 其合成的总产率为 40%。 质谱和色谱分析检测结果如表 2所示。 The whole crude product was dissolved in a 20% acetic acid solution, and the solution was passed through a Sephadex G25 cross-linked Sephadex column (2.0 x 25 cm), and the mobile phase was a 20% acetic acid solution. The main peak was collected by an ultraviolet detector, and then freeze-dried to obtain 116.2 mg of a white powder after desalting. The above-mentioned desalted peptide compound was separated and purified by reverse-phase high performance liquid chromatography (HPLC) C 18 column (XBridgeTM BEH 130 C 18 , 19 mm×250 mm). After separation, the main peak of the sample was collected and lyophilized to obtain a white pure peptide solid. Powder 82.2 mg o The purity of the purified BN-9 sample was above 98%, and the total yield of the synthesis was 40%. The results of mass spectrometry and chromatographic analysis are shown in Table 2.
表 2 BN-9的质谱和色谱分析检测结果  Table 2 Mass spectrometry and chromatographic analysis results of BN-9
分析色谱的保留时间 高分辨质谱 (MD-TOF-MS)  Analytical chromatographic retention time High Resolution Mass Spectrometry (MD-TOF-MS)
HPLC tR (min) m/z (MH+) HPLC t R (min) m/z (MH + )
肽样品  Peptide sample
体系 1 体系 2 计算值 检测值  System 1 system 2 calculated value detected value
BN-9 16.335 17.277 1112.5641 1112.5655 注： 体系 1: 梯度洗脱体系 1为： 10-100% 乙腈 /水 (0.05% TFA) (30 分钟完成)， 流速 为： l mL/min， 检测波长为 220 nm， 分析色谱柱为： Delta Pak C18, 5 μιη, 150x3.9 mm ; 体系 2: 梯度洗脱体系 2为： 10-80%乙腈 /水 (0.05% TFA) (30 分钟完成)， 流速为： 1 mL/min, 检测波长为 220 nm， 分析色谱柱为： Delta Pak C18, 5 μιη, 150x3.9 mm。 BN-9 16.335 17.277 1112.5641 1112.5655 Note: System 1: Gradient elution system 1 is: 10-100% acetonitrile/water (0.05% TFA) (completed in 30 minutes), flow rate: l mL/min, detection wavelength 220 nm Analytical column: Delta Pak C 18 , 5 μιη, 150×3.9 mm ; System 2: Gradient elution system 2: 10-80% acetonitrile/water (0.05% TFA) (completed in 30 minutes), flow rate: 1 mL/min, detection wavelength is 220 nm, analytical column: Delta Pak C 18 , 5 μιη, 150 x 3.9 mm.
通过质谱结果分析说明， 所合成的肽化合物 BN-9与设计的化合物结构一致。色谱检测 结果表明， 所合成的肽化合物 BN-9在两种不同体系的梯度洗脱时， 其保留时间分别为 16.335禾口 17.277分钟。  Analysis by mass spectrometry indicated that the synthesized peptide compound BN-9 was identical in structure to the designed compound. The chromatographic results showed that the retention time of the synthesized peptide compound BN-9 was 16.335 and 17.277 minutes, respectively, when eluted by gradients of two different systems.

Claims

权利要求书 种基于阿片肽 Biphalin和 NPFF的嵌合肽， 其结构式如下: Claims A chimeric peptide based on the opioid peptide Biphalin and NPFF has the following structural formula:
Figure imgf000014_0001
Figure imgf000014_0001
2、 如权利要求 1所述基于阿片肽 Biphalin和 NPFF的嵌合肽的合成方法， 包括以下工艺步骤： 2. A method for synthesizing a chimeric peptide based on opioid peptide Biphalin and NPFF according to claim 1, comprising the following process steps:
( 1) 树脂预处理： 将 Rink-Amide-MBHA树脂在二氯甲烷中搅拌 30-40 min， 使树脂充分溶胀后减压抽干溶剂；  (1) Resin pretreatment: Rink-Amide-MBHA resin was stirred in dichloromethane for 30-40 min, the resin was sufficiently swollen, and the solvent was drained under reduced pressure;
( 2) 脱除 Fmoc保护： 将溶胀、 抽干溶剂后的树脂在体积浓度 18-25 %的六 氢吡啶 /DMF溶液中，搅拌 3-10min后抽干，重复 2-3次；再加入体积浓度 18-25 % 的六氢吡啶 /DMF溶液，搅拌 10-15min，使 Fmoc基团脱除完全，然后抽干溶剂； 最后用 DMF洗涤除净六氢吡啶， 得到脱除 Fmoc基团保护的树脂；  (2) Removal of Fmoc protection: The resin after swelling and solvent extraction is stirred in a volume concentration of 18-25% in a solution of hexahydropyridine/DMF, stirred for 3-10 minutes, then drained, repeated 2-3 times; The solution of 18-25% hexahydropyridine/DMF is stirred for 10-15 min to completely remove the Fmoc group, and then the solvent is drained. Finally, the dihydropyridine is removed by washing with DMF to obtain a resin which is protected by Fmoc group. ;
( 3 ) 縮合： 依次将 /V-a-Fmoc保护基团氨基酸、 Λ/-羟基苯并三氮唑、 0-苯并 三氮唑 -Λ/,Λ/,Λ/',Λ/'-四甲基脲-六氟磷酸盐完全溶解于 DMF中，再加入二异丙基乙胺 后混匀得混合溶液； 然后在氩气保护下， 将脱除 Fmoc基团保护的树脂加入所述 混合溶液中搅拌反应 40-60min， 抽干溶剂； 用 DMF 重复洗涤除去未反应的 A/-a-Fmoc保护基团氨基酸、 Λ/-羟基苯并三氮唑、 0-苯并三氮唑 -Λ/,Λ/,Λ/',Λ/'-四甲基 脲-六氟磷酸盐；  (3) Condensation: The /Va-Fmoc protecting group amino acid, Λ/-hydroxybenzotriazole, 0-benzotriazole-Λ/, Λ/, Λ/', Λ/'-four The urea-hexafluorophosphate is completely dissolved in DMF, and then mixed with diisopropylethylamine to obtain a mixed solution; then the Fmoc group-protected resin is added to the mixed solution under argon protection. Stir the reaction for 40-60 min, drain the solvent; repeat washing with DMF to remove the unreacted A/-a-Fmoc protecting group amino acid, Λ/-hydroxybenzotriazole, 0-benzotriazole-Λ/, Λ/,Λ/',Λ/'-tetramethylurea-hexafluorophosphate;
所述 A/-a-Fmoc 保护基团氨基酸、 Λ/-羟基苯并三氮唑、 0-苯并三氮唑 -Λ/,Λ/,Λ/',Λ/'-四甲基脲-六氟磷酸盐的用量分别为脱除 Fmoc基团保护的树脂摩尔量 的 2-5倍；  The A/-a-Fmoc protecting group amino acid, Λ/-hydroxybenzotriazole, 0-benzotriazole-oxime/, Λ/, Λ/', Λ/'-tetramethylurea- The amount of hexafluorophosphate is 2-5 times of the molar amount of the resin protected by the Fmoc group;
所述二异丙基乙胺的用量为脱除 Fmoc基团保护的树脂摩尔量的 4-10倍； The diisopropylethylamine is used in an amount of 4 to 10 times the molar amount of the resin protected by the Fmoc group;
(4) 肽链的延长： 重复步骤 （2)、 ( 3 ) 8 次， 按照嵌合肽结构由 C-末端向 N-末端的顺序， 依次将带有 /V-a-Fmoc保护基团氨基酸逐个縮合至树脂上， 直至 完成所有氨基酸残基的縮合， 得到肽树脂； (4) Extension of the peptide chain: repeat steps (2), (3) 8 times, according to the structure of the chimeric peptide from the C-terminus In the order of the N-terminus, the amino acids having the /Va-Fmoc protecting group are sequentially condensed onto the resin one by one until the condensation of all the amino acid residues is completed to obtain a peptide resin;
( 5 ) 肽链从树脂上的切割： 按照步骤 （2) 的方法将肽树脂最后一个氨基酸 的 Fmoc基团完全脱除； 用 DCM、 MeOH交替洗涤树脂， 充分抽干溶剂后， 按每 克肽树脂加入 10-25ml的切割剂， 于室温下切割反应 1.5-5小时； 过滤， 滤液在 不高于 37°C的条件下充分减压旋干， 然后用不高于 -10°C的***中析出沉淀； 静 置后先去除上清的***， 再用水充分溶解后用分液漏斗除去***相， 水相经冷冻 干燥， 得白色粗肽固体粉末；  (5) cleavage of the peptide chain from the resin: completely remove the Fmoc group of the last amino acid of the peptide resin according to the method of the step (2); wash the resin alternately with DCM and MeOH, and thoroughly drain the solvent, and then per gram of the peptide The resin is added with 10-25 ml of the cutting agent, and the reaction is cut at room temperature for 1.5-5 hours; filtered, and the filtrate is fully dried under reduced pressure at not higher than 37 ° C, and then used in diethyl ether not higher than -10 ° C. Precipitating the precipitate; after standing, the diethyl ether of the supernatant is removed first, and then the mixture is sufficiently dissolved in water, and then the ether phase is removed by a separatory funnel, and the aqueous phase is freeze-dried to obtain a white crude peptide solid powder;
( 6) 粗肽的脱盐和纯化： 以体积浓度 10-20%的乙酸溶液为流动相， 将粗肽 通过 Sephadex G25交联葡聚糖凝胶柱脱盐， 利用紫外检测仪收集主峰后冷冻干燥， 得到脱盐的肽化合物； 再利用反向高效液相色谱柱进行分离纯化， 收集主峰， 冷 冻干燥后得到白色的纯肽固体粉末。  (6) Desalting and Purification of Crude Peptides: The crude peptide was desalted by a Sephadex G25 cross-linked Sephadex column using a 10-20% acetic acid solution as a mobile phase, and the main peak was collected by a UV detector and then freeze-dried. The desalted peptide compound was obtained; and then separated and purified by reverse-phase high performance liquid chromatography column, the main peak was collected, and lyophilized to obtain a white pure peptide solid powder.
3、 如权利要求 1所述基于阿片肽 Biphalin和 NPFF的嵌合肽的合成方法， 其 特征在于： 步骤 （5 )所述切割剂是由三氟乙酸、 三异丙基硅烷、 水以 95:2.5:2.5 的体积比混合形成的溶剂。  3. The method for synthesizing a chimeric peptide based on opioid peptide Biphalin and NPFF according to claim 1, wherein: the cleavage agent is prepared from trifluoroacetic acid, triisopropylsilane and water at 95: 2.5:2.5 by volume of solvent formed by mixing.
4、如权利要求 1所述基于阿片肽 Biphalin和 NPFF的嵌合肽在制备镇痛药物 中的应用。  4. Use of an opioid peptide Biphalin and NPFF based chimeric peptide according to claim 1 for the preparation of an analgesic medicament.
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